甜菊自交胚挽救及其自交S_1代的主要性状分析
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摘要
对甜菊(Stevia rebaudiana Bertoni)品种‘中山5号’(‘Zhongshan No. 5’)、‘中山6号’(‘Zhongshan No. 6’)、‘中山8号’(‘Zhongshan No. 8’)、‘江甜2号’(‘Jiangtian No. 2’)、‘谱星1号’(‘PC Star No. 1’)和‘守田3号’(‘Morita No. 3’)自交授粉后的有胚率进行了统计,进一步筛选出‘中山8号’的胚挽救方法,并对‘中山8号’自交S1代植株的主要性状进行了分析。结果表明:甜菊不同品种间的有胚率差异明显,其中,自交授粉后5 d‘中山8号’的有胚率最高(16. 0%),自交授粉后10 d以上6个品种的有胚率基本为0. 0%。随自交授粉后天数的增加,‘中山8号’胚珠和幼胚在MS培养基、愈伤诱导培养基(添加2. 0 mg·m L-16-BA和2. 0 mg·m L-1NAA的MS培养基)和分化诱导培养基(添加2. 0 mg·m L-16-BA和0. 2 mg·m L-1NAA的MS培养基)上的萌发时间逐渐缩短,萌发率逐渐升高。自交授粉后7～9 d‘中山8号’幼胚在MS培养基上的萌发率高达80%以上。短日照处理下,‘中山8号’自交S1代植株间现蕾期的差异较大,短日照处理10～46 d现蕾,其中,短日照处理15 d现蕾的植株最多,占植株总数的16. 5%。现蕾期‘中山8号’103个自交S1代植株叶片中莱鲍迪苷A(RA)、甜菊苷(ST)、莱鲍迪苷C(RC)、杜尔可苷A(DA)和总甜菊糖苷(TSG)的含量及RA、ST、RC和DA含量占TSG含量的比例的变化范围均较大,RA、ST、RC、DA和TSG的含量分别为0. 26%～11. 43%、0. 36%～6. 45%、1. 52%～9. 18%、0. 05%～1. 40%和5. 80%～16. 90%,RA、ST、RC和DA含量占TSG含量的比例分别为3. 19%～72. 06%、3. 20%～46. 98%、12. 59%～74. 27%和0. 53%～12. 33%。在‘中山8号’及其103个自交S1代植株中,‘中山8号’和9个自交S1代植株为高RA型,7个自交S1代植株为高RA-RC型,11个自交S1代植株为高RC型,1个自交S1代植株为高RA-RC-DA型。上述研究结果表明:胚挽救技术可克服甜菊自交不亲和受精后障碍,获得的自交S1代植株的现蕾期和甜菊糖苷含量变化范围较大,为甜菊的种质创制和新品种培育提供了有效途径。
Embryo rate of cultivar ‘Zhongshan No. 5',‘Zhongshan No. 6',‘Zhongshan No. 8',‘Jiangtian No. 2',‘PC Star No. 1',and‘Morita No. 3'of Stevia rebaudiana Bertoni after self-crossing pollination was compared,and furthermore,embryo rescue method of ‘Zhongshan No. 8'was screened.The main characters of self-crossing S1 progeny plants of ‘Zhongshan No. 8'were also analyzed. The results show that there is an obvious difference in embryo rate among different cultivars of S. rebaudiana,in which,embryo rate of ‘Zhongshan No. 8'is the highest(16. 0%) after self-crossing pollination for5 d,and those of six cultivars are basically 0. 0% after self-crossing pollination for longer than 10 d. With the increase of days after self-crossing pollination,germination times of ovule and embryo of ‘ZhongshanNo. 8'are gradually shortened on MS medium,callus induction medium(MS medium added with 2. 0 mg·m L-16-BA and 2. 0 mg·m L-1 NAA),and differentiation induction medium(MS medium added with 2. 0 mg · m L-16-BA and 0. 2 mg · m L-1 NAA),and their germination rates gradually increase.Germination rate of embryo of ‘Zhongshan No. 8 ' on MS medium is above 80% after self-crossing pollination for 7-9 d. Under short-day treatment,there is an obvious difference in budding stage of selfcrossing S1 progeny plants of ‘Zhongshan No. 8',and bud appears after short-day treatment for 10-46 d,in which,number of budding plants is the biggest after short-day treatment for 15 d,accounting for16. 5% of number of total plants. Variation ranges of contents of rebaudioside A(RA),stevioside(ST),rebaudioside C(RC),dulcoside A(DA) and total steviol glycosides(TSG),and percentages of contents of RA,ST,RC and DA to TSG content are relatively large in leaves of 103 self-crossing S1 progeny plants of ‘Zhongshan No. 8'at budding stage. Contents of RA,ST,RC,DA,and TSG are0. 26%-11. 43%,0. 36%-6. 45%,1. 52%-9. 18%,0. 05%-1. 40%, and 5. 80%-16. 90%,respectively,and percentages of contents of RA,ST,RC,and DA to TSG content are 3. 19%-72. 06%,3. 20%-46. 98%,12. 59%-74. 27%,and 0. 53%-12. 33%,respectively. Among ‘Zhongshan No. 8'and its 103 self-crossing S1 progeny plants,‘Zhongshan No. 8'and 9 self-crossing S1 progeny plants belong to high RA type,7 self-crossing S1 progeny plants belong to high RA-RC type,11 self-crossing S1 progeny plants belong to high RC type,and 1 self-crossing S1 progeny plant belongs to high RA-RC-DA type. It is suggested that the technique of embryo rescue can overcome post-crossing barrier of S.rebaudiana self-incompatibility,and variation ranges of budding stage and steviol glycoside content of self-crossing S1 progeny plants obtained are relatively large,which provides an effective approach for germplasm innovation and breeding of new cultivars.
Embryo rate of cultivar ‘Zhongshan No. 5',‘Zhongshan No. 6',‘Zhongshan No. 8',‘Jiangtian No. 2',‘PC Star No. 1',and‘Morita No. 3'of Stevia rebaudiana Bertoni after self-crossing pollination was compared,and furthermore,embryo rescue method of ‘Zhongshan No. 8'was screened.The main characters of self-crossing S1 progeny plants of ‘Zhongshan No. 8'were also analyzed. The results show that there is an obvious difference in embryo rate among different cultivars of S. rebaudiana,in which,embryo rate of ‘Zhongshan No. 8'is the highest(16. 0%) after self-crossing pollination for5 d,and those of six cultivars are basically 0. 0% after self-crossing pollination for longer than 10 d. With the increase of days after self-crossing pollination,germination times of ovule and embryo of ‘ZhongshanNo. 8'are gradually shortened on MS medium,callus induction medium(MS medium added with 2. 0 mg·m L-16-BA and 2. 0 mg·m L-1 NAA),and differentiation induction medium(MS medium added with 2. 0 mg · m L-16-BA and 0. 2 mg · m L-1 NAA),and their germination rates gradually increase.Germination rate of embryo of ‘Zhongshan No. 8 ' on MS medium is above 80% after self-crossing pollination for 7-9 d. Under short-day treatment,there is an obvious difference in budding stage of selfcrossing S1 progeny plants of ‘Zhongshan No. 8',and bud appears after short-day treatment for 10-46 d,in which,number of budding plants is the biggest after short-day treatment for 15 d,accounting for16. 5% of number of total plants. Variation ranges of contents of rebaudioside A(RA),stevioside(ST),rebaudioside C(RC),dulcoside A(DA) and total steviol glycosides(TSG),and percentages of contents of RA,ST,RC and DA to TSG content are relatively large in leaves of 103 self-crossing S1 progeny plants of ‘Zhongshan No. 8'at budding stage. Contents of RA,ST,RC,DA,and TSG are0. 26%-11. 43%,0. 36%-6. 45%,1. 52%-9. 18%,0. 05%-1. 40%, and 5. 80%-16. 90%,respectively,and percentages of contents of RA,ST,RC,and DA to TSG content are 3. 19%-72. 06%,3. 20%-46. 98%,12. 59%-74. 27%,and 0. 53%-12. 33%,respectively. Among ‘Zhongshan No. 8'and its 103 self-crossing S1 progeny plants,‘Zhongshan No. 8'and 9 self-crossing S1 progeny plants belong to high RA type,7 self-crossing S1 progeny plants belong to high RA-RC type,11 self-crossing S1 progeny plants belong to high RC type,and 1 self-crossing S1 progeny plant belongs to high RA-RC-DA type. It is suggested that the technique of embryo rescue can overcome post-crossing barrier of S.rebaudiana self-incompatibility,and variation ranges of budding stage and steviol glycoside content of self-crossing S1 progeny plants obtained are relatively large,which provides an effective approach for germplasm innovation and breeding of new cultivars.
引文
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[8]王淑明.柑橘纯合种质创制及早金甜橙DH系代谢与转录组分析[D].武汉:华中农业大学园艺林学学院,2016:42-48.
[9]夏晶.不同倍性百合(Lilium spp.)杂交后代胚挽救及染色体分析[D].重庆:西南大学园艺学院,2010:17-20.
[10]罗建让,张延龙,张林华.克服百合自交及杂交障碍方法的初步研究[J].西北农业学报,2007,16(4):260-263.
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[13]李辛雷,陈发棣.菊花二倍体野生种与栽培种间杂种的幼胚拯救[J].林业科学,2006,42(11):42-46.
[14]李辛雷.菊属植物自交、杂交及远缘杂种幼胚拯救研究[D].南京:南京农业大学园艺学院,2004:26-31.
[15]李辛雷,陈发棣.菊属种间杂交胚拯救过程成苗途径的初步研究[J].植物学通报,2004,21(3):337-341.
[16]顾爱侠,冯大领,轩淑欣,等.胚挽救技术在植物育种中的应用[J].河北林果研究,2010,25(1):30-33.
[17]张倩云,王莹,杜玉梁,等.植物胚拯救技术的影响因素及其应用[J].南京林业大学学报(自然科学版),2014,38(5):143-148.
[18]朱际君,汪祖华,蔡兴元.特早熟桃幼胚培养技术[J].江苏农业学报,1985,1(3):41-43.
[19]申家恒,殷华,连永权,等.甜叶菊的受精作用及胚和胚乳的早期发育[J].植物学报,1989,31(3):165-170.
[20] SINGH G,SINGH G,SINGH P,et al. Molecular dissection of transcriptional reprogramming of steviol glycosides synthesis in leaf tissue during developmental phase transitions in Stevia rebaudiana Bert[J]. Scientific Reports,2017,7(1):11835.
[21] YANG Y,HUANG S,HAN Y,et al. Environmental cues induce changes of steviol glycosides contents and transcription of corresponding biosynthetic genes in Stevia rebaudiana[J]. Plant Physiology and Biochemistry,2015,86(86C):174-180.
[22] CEUNEN S,GEUNS J M C. Spatio-temporal variation of the diterpene steviol in Stevia rebaudiana grown under different photoperiods[J]. Phytochemistry,2013,89(1):32-38.
[2] YADAV A K,SINGH S,DHYANI D,et al. A review on the improvement of stevia[Stevia rebaudiana(Bertoni)][J]. Canadian Journal of Plant Science,2011,91(1):1-27.
[3]袁鎏柳,罗庆云,陈思锐,等.聚药雄蕊植物甜叶菊花部特征与繁育特性初步研究[J].中国糖料,2018,40(3):1-6.
[4] SHARMA D R,KAUR R,KUMAR K. Embryo rescue in plants:a review[J]. Euphytica,1996,89(3):325-337.
[4]李桂英,王琳清,施巾帼.小麦与窄颖赖草远缘杂交幼胚拯救方法的探讨[J].麦类作物学报,2005,25(5):10-13.
[5]周传恩,夏光敏.小麦远缘杂交胚拯救技术[J].麦类作物学报,2005,25(3):88-92.
[6]田淑芬,温晓敏,张娜,等.无核葡萄自交胚挽救技术研究[J].中外葡萄与葡萄酒,2017(4):25-27.
[7]程琳琳,王跃进,张剑侠,等.胚挽救技术进行无核葡萄及三倍体新种质创制[J].热带作物学报,2012,33(9):1584-1589.
[8]王淑明.柑橘纯合种质创制及早金甜橙DH系代谢与转录组分析[D].武汉:华中农业大学园艺林学学院,2016:42-48.
[9]夏晶.不同倍性百合(Lilium spp.)杂交后代胚挽救及染色体分析[D].重庆:西南大学园艺学院,2010:17-20.
[10]罗建让,张延龙,张林华.克服百合自交及杂交障碍方法的初步研究[J].西北农业学报,2007,16(4):260-263.
[11]甘欣,魏臻武,武自念,等.多叶苜蓿自交S1代的胚挽救培养[J].草业科学,2011,28(2):226-230.
[12]陈雪鹃,孙明,菅琳,等.部分菊属植物及其近缘种的胚拯救与杂种鉴定[J].东北林业大学学报,2014,42(7):74-79,86.
[13]李辛雷,陈发棣.菊花二倍体野生种与栽培种间杂种的幼胚拯救[J].林业科学,2006,42(11):42-46.
[14]李辛雷.菊属植物自交、杂交及远缘杂种幼胚拯救研究[D].南京:南京农业大学园艺学院,2004:26-31.
[15]李辛雷,陈发棣.菊属种间杂交胚拯救过程成苗途径的初步研究[J].植物学通报,2004,21(3):337-341.
[16]顾爱侠,冯大领,轩淑欣,等.胚挽救技术在植物育种中的应用[J].河北林果研究,2010,25(1):30-33.
[17]张倩云,王莹,杜玉梁,等.植物胚拯救技术的影响因素及其应用[J].南京林业大学学报(自然科学版),2014,38(5):143-148.
[18]朱际君,汪祖华,蔡兴元.特早熟桃幼胚培养技术[J].江苏农业学报,1985,1(3):41-43.
[19]申家恒,殷华,连永权,等.甜叶菊的受精作用及胚和胚乳的早期发育[J].植物学报,1989,31(3):165-170.
[20] SINGH G,SINGH G,SINGH P,et al. Molecular dissection of transcriptional reprogramming of steviol glycosides synthesis in leaf tissue during developmental phase transitions in Stevia rebaudiana Bert[J]. Scientific Reports,2017,7(1):11835.
[21] YANG Y,HUANG S,HAN Y,et al. Environmental cues induce changes of steviol glycosides contents and transcription of corresponding biosynthetic genes in Stevia rebaudiana[J]. Plant Physiology and Biochemistry,2015,86(86C):174-180.
[22] CEUNEN S,GEUNS J M C. Spatio-temporal variation of the diterpene steviol in Stevia rebaudiana grown under different photoperiods[J]. Phytochemistry,2013,89(1):32-38.