大麦单粒种子起源的小孢子培养再生频率研究
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摘要
为提高单株小孢子培养的再生效率,以大麦品种花30为材料,在人工气候室研究了离体穗、离体小花预处理时间和诱导培养基中无机氮、有机氮含量以及添加PEG对愈伤组织产量、绿苗产量和再生能力的影响。结果表明,离体穗5℃处理19 d的愈伤组织和绿苗产量及再生能力均优于32 d的,离体小花预处理1～2 d可促进愈伤组织的形成和绿苗的分化;降低无机氮浓度[KNO_3降至707.5 mg·L~(-1),(NH_4)_2SO_4降至115.75 mg·L~(-1)]可提高愈伤组织和绿苗产量及再生能力;提高有机氮浓度(谷氨酰胺从400 mg·L~(-1)提高到1 600 mg·L~(-1),水解酪蛋白从100 mg·L~(-1)提高到400 mg·L~(-1))可增加小孢子愈伤组织和绿苗产量;培养基中添加4%PEG可提高小孢子愈伤组织产量和质量。
In order to improve the regeneration frequency of isolated microspores derived from a single-seed of barley,a single donor plant grown in chamber for barley cultivar Hua 30,and the effects of the period of pretreatment on young spikes and floret,the content of inorganic and organic nitrogen in induction medium,and the addition of PEG on the formation of callus and green plants were studied. The results showed that the pretreatment of 19 d at 5℃ on young spikes worked better than the pretreatment of 32 d. The pretreatment on floret for 1-2 d benefited the formation of callus and green plants. Low level of inorganic nitrogen in induction medium[KNO_3 reduced to 707.5 mg·L~(-1) and(NH_4)_2SO_4 reduced to 115.75 mg·L~(-1)] improved the production of both callus and green plants. The elevation of organic nitrogen level(casein glutamine increased from 400 mg·L~(-1) to 1 600 mg·L~(-1),and hydrolysate increased from 100 mg·L~(-1) to 400 mg·L~(-1)) promoted the callus formation and green plant regeneration. In addition,the supplement of 4% PEG also improved the quantity and quality of callus formation.
In order to improve the regeneration frequency of isolated microspores derived from a single-seed of barley,a single donor plant grown in chamber for barley cultivar Hua 30,and the effects of the period of pretreatment on young spikes and floret,the content of inorganic and organic nitrogen in induction medium,and the addition of PEG on the formation of callus and green plants were studied. The results showed that the pretreatment of 19 d at 5℃ on young spikes worked better than the pretreatment of 32 d. The pretreatment on floret for 1-2 d benefited the formation of callus and green plants. Low level of inorganic nitrogen in induction medium[KNO_3 reduced to 707.5 mg·L~(-1) and(NH_4)_2SO_4 reduced to 115.75 mg·L~(-1)] improved the production of both callus and green plants. The elevation of organic nitrogen level(casein glutamine increased from 400 mg·L~(-1) to 1 600 mg·L~(-1),and hydrolysate increased from 100 mg·L~(-1) to 400 mg·L~(-1)) promoted the callus formation and green plant regeneration. In addition,the supplement of 4% PEG also improved the quantity and quality of callus formation.
引文
[1]董静,邬飞波,张国平.麦类作物小孢子培养研究进展[J].麦类作物学报,2005,25(2):105. DONG J,WU F B,ZHANG G P.Advance on isolated microspore culture of Triticeae crops [J].Journal of Triticeae Crops,2005,25(2):105.
[2]赵爱菊,高增玉,李亚军,等.禾谷类作物的小孢子培养[J].河北农业科学,2008,12(4):73. ZHAO A J,GAO Z Y,LI Y J,et al.Study on methods of cereal crops microspore culture [J].Journal of Hebei Agricultural Sciences,2008,12(4):73.
[3]陆瑞菊,黄剑华,何南扬,等.应用小孢子离体培养技术培育大麦新品系[J].麦类作物学报,2002,22(4):89. LU R J,HUANG J H,HE N Y,et al.Development of new barley strain by microspore culture technique [J].Journal of Triticeae Crops,2002,22(4):89.
[4]LU R J,WANG Y F,SUN Y F,et al.Improvement of isolated microspore culture of barley(Hordeum vulgare L.):The effect of floret co-culture [J].Plant Cell,Tissue and Organ Culture,2008,93(1):22.
[5]郭桂梅,何婷,刘成洪,等.大麦单株小孢子植株再生及胁迫培养研究[J].植物生理学报,2015,51(12):2202. GUO G M,HE T,LIU C H,et al.Regeneration from single plant via microspore culture and culture response to salt stress [J].Plant Physiology Journal,2015,51(12):2202.
[6]黄剑华,陆瑞菊,王亦菲,等.提高迟钝型大麦花培反应率的因素[J].上海农业学报,2000,16(4):15. HUANG J H,LU R J,WANG Y F,et al.Studies on factors improving anther culture response of low responding genotypes in barley [J].Acta Agriculturae Shanghai,2000,16(4):15.
[7]陆瑞菊.大麦游离小孢子培养技术的优化及单倍体耐盐、耐低氮胁迫筛选体系的建立[D].南京:南京农业大学,2012:9-34. LU R J.System optimization of isolated microspore culture and screening of stress tolerances to salt and low nitrogen in haploid barley [D].Nanjing:Nanjing Agricultural University,2012:9-34.
[8]何婷,郭桂梅,陈志伟,等.大麦小孢子再生植株气孔保卫细胞长度与倍性的相关性[J].麦类作物学报,2014,34(2):175. HE T,GUO G M,CHEN Z W,et al.Relationship between the stomatal guard cell length and ploidy level in barley microspore regenerated plantlets [J].Journal of Triticeae Crops,2014,34(2):175.
[9]郭桂梅.水稻和大麦游离小孢子培养条件的优化[D].上海:上海海洋大学,2013:29-49. GUO G M.Optimization on isolated microspore culture for rice and barley [D].Shanghai:Shanghai Ocean University,2013:29-49.
[10]陆瑞菊,黄剑华,孙月芳,等.秋水仙碱对大麦离体培养小孢子存活与成苗的影响[J].植物生理学报,2001,27(2):135. LU R J,HUANG J H,SUN Y F,et al.The effect of colchicine on survival and regeneration of isolated microspores in barley [J].Plant Physiology Journal,2001,27(2):135.
[11]郭桂梅,何婷,高润红,等.预处理和培养温度对陆稻游离小孢子愈伤组织诱导及分化的影响[J].植物生理学报,2014,50(12):1760. GUO G M,HE T,GAO R H,et al.Effects of pretreatments and culture temperature on callus induction and differentiation in upland rice microspore culture [J].Plant Physiology Journal,2014,50(12):1760.
[12]郭向荣,景建康,胡含.大麦不同基因型游离小孢子的直接培养再生及培养体系的优化[J].遗传学报,1997,24(6):508. GUO X R,JING J K,HU H.Plant regeneration and optimization of culture system in direct culture of isolated microspores from different genotypes of barley [J].Journal of Genetics and Genomics,1997,24(6):508.
[13]黄斌.大麦花药培养中低温预处理对花粉愈伤组织形成的影响[J].植物学报,1985,27(3):441. HUANG B.Influence of cold pretreatment on the production of pollen callus in anther cultures of barley [J].Acta Botanica Sinica,1985,27(3):441.
[14]POWELL W.The influence of genotype and temperature pre-treatment on anther culture response in barley(Hordeum vulgare L.) [J].Plant Cell,Tissue and Organ Culture,1988,12(3):295.
[15]黄剑华,陆瑞菊,王亦菲,等.低温预处理对大麦花药内源游离氨基酸和多胺水平的影响[J].中国农业科学,2001,34(6):630. HUANG J H,LU R J,WANG Y F,et al.Effects of cool pretreatment on level of amino acid and polyamine content in barley anthers [J].Scientia Agricultura Sinica,2001,34(6):627.
[16]WEI Z M,KYO M,HARADA H.Callus formation and plant regeneration through direct culture of isolated pollen of Hordeum vulgare cv.‘Sabarlis’ [J].Theoretical and Applied Genetics,1986,72(2):253.
[17]陆瑞菊,高润红,郭桂梅,等.青稞的小孢子离体培养和植株再生[J].植物生理学报,2014,50(12):1765. LU R J,GAO R H,GUO G M,et al.Microspore culture and plantlet regeneration of highland barley(Hordeum vulgare var.nudum) [J].Plant Physiology Journal,2014,50(12):1765.
[18]郭桂梅,高润红,卜姝明,等.预处理对水稻小孢子诱导愈伤组织产量和绿苗分化的影响[J].上海农业学报,2014,30(5):52. GUO G M,GAO R H,BU S M,et al.Effects of pretreatments on callus induction and green plantlet differentiation in rice microspore culture in vitro [J].Acta Agriculturae Shanghai,2014,30(5):52.
[19]陆瑞菊,孙月芳,王亦菲,等.提高大麦游离小孢子培养反应的研究[J].大麦科学,2004(2):16. LU R J,SUN Y F,WANG Y F,et al.Studies on improving response of microspore culture in barley [J].Barley Science,2004(2):16.
[20]王宏芝.小麦游离小孢子培养研究进展[J].麦类作物学报,2002,22(1):92. WANG H Z.The research progress on embryogenesis in isolated wheat microspores [J].Journal of Triticeae Crops,2002,22(1):92.
[21]王培,陈玉蓉.C17培养基在花药培养中应用的研究[J].植物学报,1986,28(1):39. WANG P,CHEN Y R.A study on the application of C17 medium for anther culture [J].Acta Botanica Sinica,1986,28(1):39.
[22]吕学莲,白海波,董建力,等.培养基及高温暗培养时间对小麦花药培养的影响[J].种子,2015,34(6):85. Lü X L,BAI H B,DONG J L,et al.Effects of medium and the darkness culture time of high-temperature on wheat anther culture [J].Seed,2015,34(6):85.
[23]OLSEN F L.Induction of microspore embryogenesis in cultured anthers of Hordeum vulgare.The effects of ammonium nitrate,glutamine and asparagine as nitrogen sources [J].Carlsberg Research Communications,1987,52(6):402.
[24]LU R J,CHEN Z W,GAO R H,et al.Genotypes-independent optimization of nitrogen supply for isolated microspore cultures in barley [J].BioMed Research International,2016,1801646:2.
[25]RITALA A,MANNONEN L,OKSMAN-CALDENTEY K M.Factors affecting the regeneration capacity of isolated barley microspores(Hordeum vulgare L.) [J].Plant Cell Reports,2001,20(5):406.
[26]SHAHSAVARI E.Impact of tryptophan and glutamine on the tissue culture of upland rice [J].Plant Soil Environment,2011,57(1):8.
[27]PAWAR B,KALE P,BAHURUPE J,et al.Proline and glutamine improve in vitro callus induction and subsequent shooting in rice [J].Rice Science,2015,22:285.
[28]SRISKANDARAJAH S,SAMERI M,LERCETEAU-KO HLER E,et al.Increased recovery of green doubled haploid plants from barley anther culture [J].Crop Science,2015,55:2809.
[29]刘桂茹,葛淑俊,王静华,等.PEG对小麦花药出愈率的影响[J].河北农业大学学报,2001,24(4):27. LIU G R,GE S J,WANG J H,et al.The effect of the PEG on the initiating rate of the anther culture in wheat [J].Journal of Agricultural University of Hebei,2001,24(4):27.
[30]王炜,王剑虹,罗俊杰,等.干旱胁迫对小麦花药培养的影响[J].麦类作物学报,2016,36(5):644. WANG W,WANG J H,LUO J J,et al.Effect of drought stress on wheat anther culture [J].Journal of Triticeae Crops,2016,36(5):644.
[31]KAO K N,SALEEM M,ABRAMS S,et al.Culture conditions for induction of green plants from barley microspores by anther culture methods [J].Plant Cell Reports,1991,9:598.
[2]赵爱菊,高增玉,李亚军,等.禾谷类作物的小孢子培养[J].河北农业科学,2008,12(4):73. ZHAO A J,GAO Z Y,LI Y J,et al.Study on methods of cereal crops microspore culture [J].Journal of Hebei Agricultural Sciences,2008,12(4):73.
[3]陆瑞菊,黄剑华,何南扬,等.应用小孢子离体培养技术培育大麦新品系[J].麦类作物学报,2002,22(4):89. LU R J,HUANG J H,HE N Y,et al.Development of new barley strain by microspore culture technique [J].Journal of Triticeae Crops,2002,22(4):89.
[4]LU R J,WANG Y F,SUN Y F,et al.Improvement of isolated microspore culture of barley(Hordeum vulgare L.):The effect of floret co-culture [J].Plant Cell,Tissue and Organ Culture,2008,93(1):22.
[5]郭桂梅,何婷,刘成洪,等.大麦单株小孢子植株再生及胁迫培养研究[J].植物生理学报,2015,51(12):2202. GUO G M,HE T,LIU C H,et al.Regeneration from single plant via microspore culture and culture response to salt stress [J].Plant Physiology Journal,2015,51(12):2202.
[6]黄剑华,陆瑞菊,王亦菲,等.提高迟钝型大麦花培反应率的因素[J].上海农业学报,2000,16(4):15. HUANG J H,LU R J,WANG Y F,et al.Studies on factors improving anther culture response of low responding genotypes in barley [J].Acta Agriculturae Shanghai,2000,16(4):15.
[7]陆瑞菊.大麦游离小孢子培养技术的优化及单倍体耐盐、耐低氮胁迫筛选体系的建立[D].南京:南京农业大学,2012:9-34. LU R J.System optimization of isolated microspore culture and screening of stress tolerances to salt and low nitrogen in haploid barley [D].Nanjing:Nanjing Agricultural University,2012:9-34.
[8]何婷,郭桂梅,陈志伟,等.大麦小孢子再生植株气孔保卫细胞长度与倍性的相关性[J].麦类作物学报,2014,34(2):175. HE T,GUO G M,CHEN Z W,et al.Relationship between the stomatal guard cell length and ploidy level in barley microspore regenerated plantlets [J].Journal of Triticeae Crops,2014,34(2):175.
[9]郭桂梅.水稻和大麦游离小孢子培养条件的优化[D].上海:上海海洋大学,2013:29-49. GUO G M.Optimization on isolated microspore culture for rice and barley [D].Shanghai:Shanghai Ocean University,2013:29-49.
[10]陆瑞菊,黄剑华,孙月芳,等.秋水仙碱对大麦离体培养小孢子存活与成苗的影响[J].植物生理学报,2001,27(2):135. LU R J,HUANG J H,SUN Y F,et al.The effect of colchicine on survival and regeneration of isolated microspores in barley [J].Plant Physiology Journal,2001,27(2):135.
[11]郭桂梅,何婷,高润红,等.预处理和培养温度对陆稻游离小孢子愈伤组织诱导及分化的影响[J].植物生理学报,2014,50(12):1760. GUO G M,HE T,GAO R H,et al.Effects of pretreatments and culture temperature on callus induction and differentiation in upland rice microspore culture [J].Plant Physiology Journal,2014,50(12):1760.
[12]郭向荣,景建康,胡含.大麦不同基因型游离小孢子的直接培养再生及培养体系的优化[J].遗传学报,1997,24(6):508. GUO X R,JING J K,HU H.Plant regeneration and optimization of culture system in direct culture of isolated microspores from different genotypes of barley [J].Journal of Genetics and Genomics,1997,24(6):508.
[13]黄斌.大麦花药培养中低温预处理对花粉愈伤组织形成的影响[J].植物学报,1985,27(3):441. HUANG B.Influence of cold pretreatment on the production of pollen callus in anther cultures of barley [J].Acta Botanica Sinica,1985,27(3):441.
[14]POWELL W.The influence of genotype and temperature pre-treatment on anther culture response in barley(Hordeum vulgare L.) [J].Plant Cell,Tissue and Organ Culture,1988,12(3):295.
[15]黄剑华,陆瑞菊,王亦菲,等.低温预处理对大麦花药内源游离氨基酸和多胺水平的影响[J].中国农业科学,2001,34(6):630. HUANG J H,LU R J,WANG Y F,et al.Effects of cool pretreatment on level of amino acid and polyamine content in barley anthers [J].Scientia Agricultura Sinica,2001,34(6):627.
[16]WEI Z M,KYO M,HARADA H.Callus formation and plant regeneration through direct culture of isolated pollen of Hordeum vulgare cv.‘Sabarlis’ [J].Theoretical and Applied Genetics,1986,72(2):253.
[17]陆瑞菊,高润红,郭桂梅,等.青稞的小孢子离体培养和植株再生[J].植物生理学报,2014,50(12):1765. LU R J,GAO R H,GUO G M,et al.Microspore culture and plantlet regeneration of highland barley(Hordeum vulgare var.nudum) [J].Plant Physiology Journal,2014,50(12):1765.
[18]郭桂梅,高润红,卜姝明,等.预处理对水稻小孢子诱导愈伤组织产量和绿苗分化的影响[J].上海农业学报,2014,30(5):52. GUO G M,GAO R H,BU S M,et al.Effects of pretreatments on callus induction and green plantlet differentiation in rice microspore culture in vitro [J].Acta Agriculturae Shanghai,2014,30(5):52.
[19]陆瑞菊,孙月芳,王亦菲,等.提高大麦游离小孢子培养反应的研究[J].大麦科学,2004(2):16. LU R J,SUN Y F,WANG Y F,et al.Studies on improving response of microspore culture in barley [J].Barley Science,2004(2):16.
[20]王宏芝.小麦游离小孢子培养研究进展[J].麦类作物学报,2002,22(1):92. WANG H Z.The research progress on embryogenesis in isolated wheat microspores [J].Journal of Triticeae Crops,2002,22(1):92.
[21]王培,陈玉蓉.C17培养基在花药培养中应用的研究[J].植物学报,1986,28(1):39. WANG P,CHEN Y R.A study on the application of C17 medium for anther culture [J].Acta Botanica Sinica,1986,28(1):39.
[22]吕学莲,白海波,董建力,等.培养基及高温暗培养时间对小麦花药培养的影响[J].种子,2015,34(6):85. Lü X L,BAI H B,DONG J L,et al.Effects of medium and the darkness culture time of high-temperature on wheat anther culture [J].Seed,2015,34(6):85.
[23]OLSEN F L.Induction of microspore embryogenesis in cultured anthers of Hordeum vulgare.The effects of ammonium nitrate,glutamine and asparagine as nitrogen sources [J].Carlsberg Research Communications,1987,52(6):402.
[24]LU R J,CHEN Z W,GAO R H,et al.Genotypes-independent optimization of nitrogen supply for isolated microspore cultures in barley [J].BioMed Research International,2016,1801646:2.
[25]RITALA A,MANNONEN L,OKSMAN-CALDENTEY K M.Factors affecting the regeneration capacity of isolated barley microspores(Hordeum vulgare L.) [J].Plant Cell Reports,2001,20(5):406.
[26]SHAHSAVARI E.Impact of tryptophan and glutamine on the tissue culture of upland rice [J].Plant Soil Environment,2011,57(1):8.
[27]PAWAR B,KALE P,BAHURUPE J,et al.Proline and glutamine improve in vitro callus induction and subsequent shooting in rice [J].Rice Science,2015,22:285.
[28]SRISKANDARAJAH S,SAMERI M,LERCETEAU-KO HLER E,et al.Increased recovery of green doubled haploid plants from barley anther culture [J].Crop Science,2015,55:2809.
[29]刘桂茹,葛淑俊,王静华,等.PEG对小麦花药出愈率的影响[J].河北农业大学学报,2001,24(4):27. LIU G R,GE S J,WANG J H,et al.The effect of the PEG on the initiating rate of the anther culture in wheat [J].Journal of Agricultural University of Hebei,2001,24(4):27.
[30]王炜,王剑虹,罗俊杰,等.干旱胁迫对小麦花药培养的影响[J].麦类作物学报,2016,36(5):644. WANG W,WANG J H,LUO J J,et al.Effect of drought stress on wheat anther culture [J].Journal of Triticeae Crops,2016,36(5):644.
[31]KAO K N,SALEEM M,ABRAMS S,et al.Culture conditions for induction of green plants from barley microspores by anther culture methods [J].Plant Cell Reports,1991,9:598.