不同金属离子诱导枸杞体细胞胚发生及枸杞多倍体培育研究
摘要
植物体细胞胚发生的机理研究和多倍体诱导的技术优化一直是植物分子及细胞生物学中的两个热点问题。已有的研究表明,不同价位金属离子分别对宁夏枸杞(Lycium barbarum L.)体细胞胚发生的调节效应和作用机制差异显著,但在同一条件下的比较试验严重不足,且以体细胞胚为基底的同源四倍体诱导非常少见。本研究以宁夏枸杞代表品种—宁红1号为试验材料,以不同价位的金属离子Ag~+、Ca~(2+)和Eu~(3+)的可溶性化合物按照不同浓度梯度作为外源影响因子,试验研究了三种不同外植体根基部、茎和叶切块体细胞胚发生的效率和相对生长速率;同时试验比较了体细胞胚、愈伤组织和激活种子在不同浓度梯度的秋水仙素和DMSO混合液诱导下枸杞同源四倍体发生效率,并对育成的同源四倍体进行了细胞学和形态学分析。以上两个方面为今后枸杞体细胞胚诱导的高效体系的建立和植物同源多倍体技术优化提供理论基础,为将来名贵中药材高效同质多倍体的培育、减少或避免嵌合体干扰的产生、提高诱变效率提供实践途径。主要试验结果如下:
1.枸杞无菌苗长至3-4片真叶后,将根基部、茎和叶切成小块,在MS+2,4-D0.2mg/L的诱导培养基上进行愈伤组织诱导,发现真叶被诱导得到的胚性愈伤组织长势最好、相对生长速率最高,转入无激素MS培养基时得到的体细胞胚数最多。叶愈伤组织比起根基部和茎被诱导得到的愈伤组织质地紧密,相对生长速率达到0.04g/(d·外植体)、而根基部和茎的都是0.02g/(d·外植体);转入无激素MS培养基15天时,每块胚性愈伤组织所诱导的体细胞胚数分别是根39.1个、茎42.8个、叶44.9个。
2.根基部、茎和叶为外植体诱导得到的愈伤组织在含2,4-D 0.2mg/L的MS培养基上4次继代培养后,转入含不同浓度梯度的Ag~+、Ca~(2+)和Eu~(3+)三种外源离子的MS培养基进行分化培养,发现三种离子在低浓度情况下均可促进愈伤组织的体细胞胚发生,而高浓度抑制体细胞胚的形成,且体细胞胚数在三种外植体中具有相同的变化趋势。当Ag+浓度为1mg/L时,每块叶愈伤组织形成的体细胞胚数为对照组的2倍多,Ca2+和Eu3+在其较适宜浓度下均无达到这个水平。
3.枸杞多倍体诱导:
愈伤组织:枸杞无菌苗叶片在MS+0.1mg/L6BA+0.5mg/LNAA激素条件下诱导出来的无绿色芽点、质地紧密的愈伤组织为试验材料,在0.2%秋水仙素和2%DMSO混合液处理48h时诱导率高达66.7%,且嵌合体少、同质性高,是较好的枸杞多倍体诱导的方法。
激活种子:将枸杞种子经常规消毒后浸于无菌水中,待种子露白时用0.2%秋水仙素和2%DMSO混合液浸泡24h时诱导率最高达8%,但嵌合体现象严重,多倍体的特征不稳定,表现出不同程度的回复突变。
体细胞胚:枸杞无菌苗叶片在含0.2mg/L2,4-D的MS培养基上诱导出来的愈伤组织在相同培养基上继代培养4次后,转入不含激素的MS培养基上分化10天所得到的球形胚为试验材料,在20mg/L秋水仙素和1%DMSO混合液处理24h时诱变率6%、无嵌合体现象。本文首次报道,在体细胞胚的离体条件下,通过秋水仙素处理诱导获得了枸杞同源四倍体。
4.染色体计数鉴定表明,正常二倍体植株染色体数为24,所得到的四倍体植株染色体增加一倍,为48条染色体。
5.从形态学和细胞学方面对四倍体植株和二倍体植株进行了比较。结果显示,形态上四倍体比二倍体植株根系较少,但粗壮。四倍体植株与二倍体植株在气孔大小、频度具极显著差异。在40倍镜下,四倍体气孔长度31.9μm,二倍体植株气孔长度24.3μm;四倍体宽度29.0μm,二倍体宽度23.9μm。而在10倍镜视野内,气孔频度四倍体的21.6个,二倍体的是27个。四倍体气孔叶绿体数分布范围在24—50个,其中叶绿体数为38的占17%左右;二倍体气孔包围细胞叶绿体数分布范围在12—28个,其中叶绿体数为20的占25%。四倍体和二倍体气孔叶绿体数之比约等于2。
In recent years, the study of development of plant somatic embryo and technique of polyploid induction have become two important subjects in cytobiology and molecular biology. Many researches indicated that different valent metal ions had significantly different effect on adjusting somatic embryogenesis and had not same way of regulating for Lycium barbarum L.. However, the meaningful data was not sufficient to support it under same experimental condition. For polyploid induction, using somatic embryo to induce autotetraploid was also rarely reported.
In this study, Lycium barbarum L.(ninghong 1 hao) in Ningxia was used as materials for studying induction efficiency and relative growth rate of somatic embryo with three different explants ( root, stem and leaf) and different concentration of Ag~+、Ca~(2+) and Eu~(3+) as exogenous factor.
To induce the autotetraploid, somatic embryo, callus and seed were treated with different concentration of colchicines and DMSO mixture and was induced successfully into autotetraploidy plant from all of them. The inducing efficiency was compared for the three source of explant and some cytological and morphological analysis were also done to confirm the tetraploidy.
The objective of this study was to establish efficient somatic embryo induction system, optimize the technic of inducing plant autopolyploid and provide a method of cultivating homogeneity autotetraploid without chimera. The results are as follows:
1.When axenic Lycium barbarum L. had 3-4 leaves, the pieces of root , stem and leaf were induced on MS+2,4-D0.2mg/L. The result showed that the callus from leaf had highest of growth and the largest numbers of somatic embryo on MS medium without hormones. Leaf callus was more tight than that of stem and root. The relative growth rate of leaf callus reached 0.04g/(d·explant), while stem callus was (0.02g/(d·explant)) and root callus was (0.02g/(d·explant)); After cultured for 15 days on MS medium without hormones, the numbers of somatic embryo of a piece of callus was: 39.1 (root),42.8 (stem), 44.9 (leaf).
2.After 4 generation subculture on MS+2,4-D0.2mg/L, the callus induced from root, stem and leaf were transfered to MS medium with different concentration Ag~+、Ca~(2+) and Eu~(3+) for comparing the metal ion effect on embryogenesis. The result showed that low level of Ag~+、Ca~(2+) and Eu~(3+) raised the number of the embryo, but high level reduced it. The change tendency was same for three different explants.
When the concentration of Ag~+ was 1mg/L, it raised the number of somatic embryo as 2 times as that of control group. The promoting effect of Ca~(2+) and Eu~(3+) was not as good as Ag~+ when in their best level.
3.polyploid induction
From Callus, which was induced from leaves cultured on MS+ 6-BA0.1mg/L+ NAA0.1mg/L, the tight and colourless primordium cut from this callus was treated with0.2%colchicines and2%DMSO mixture for 48h and the 66.7% polyploid induction rate was get after cultured on the original medium, more than this, little chimera to be find.
From germinated seed, which was sterilized and germinated in sterile water, the rooting seed was treated with 0.2% colchicine and 2%DMSO mixture for 24h and 8% polyploid induction rate was get after cultured on the MS medium, but the polyploid was unstable because of chimerism and reversal mutation.
From somatic embryo, the globular embryoid induced from leaf by above mentioned method was treated with 20mg/L colchicine and 1%DMSO mixture medium for 24h, 6% polyploid induction rates was get without chimerism. This is the first report for inducing autotetraploid of Lycium barbarum L. from somatic embryo.
4.Chromosome number counting assay showed that the chromosome number of normal diploid is 24 and that of the polyploid is 48. This is confirmed the tetraploidy of the polyploid get in this experiment.
5.The morphological and cytological study indicated that the tetraploid grown slowly, the green color of its leaf was deep, its stem was stronger, its root was fewer but stronger, compared to diploid.
Comparing stomata of tetraploid and diploid also gave significant difference in number and size of pore. The data is as follows:
Under light microscope with 40×, for length of pore, tetraploid (31.9um). diploid.( 24.3um), for width, tetraploid (29.0um). diploid.(23.9um).
Under light microscope with 10×, for frequency ( number of pore in one field):tetraploid (21.6). diploid.(27). For amount of chloroplasts in one stomata , tetraploid ( 24~ 50) , 17% stomata was 38; diploid (12~ 48), 25% stomata was 20. So, the amount of chloroplast in defence cell of tetraploid is as about 2 times as that of diploid.
1.枸杞无菌苗长至3-4片真叶后,将根基部、茎和叶切成小块,在MS+2,4-D0.2mg/L的诱导培养基上进行愈伤组织诱导,发现真叶被诱导得到的胚性愈伤组织长势最好、相对生长速率最高,转入无激素MS培养基时得到的体细胞胚数最多。叶愈伤组织比起根基部和茎被诱导得到的愈伤组织质地紧密,相对生长速率达到0.04g/(d·外植体)、而根基部和茎的都是0.02g/(d·外植体);转入无激素MS培养基15天时,每块胚性愈伤组织所诱导的体细胞胚数分别是根39.1个、茎42.8个、叶44.9个。
2.根基部、茎和叶为外植体诱导得到的愈伤组织在含2,4-D 0.2mg/L的MS培养基上4次继代培养后,转入含不同浓度梯度的Ag~+、Ca~(2+)和Eu~(3+)三种外源离子的MS培养基进行分化培养,发现三种离子在低浓度情况下均可促进愈伤组织的体细胞胚发生,而高浓度抑制体细胞胚的形成,且体细胞胚数在三种外植体中具有相同的变化趋势。当Ag+浓度为1mg/L时,每块叶愈伤组织形成的体细胞胚数为对照组的2倍多,Ca2+和Eu3+在其较适宜浓度下均无达到这个水平。
3.枸杞多倍体诱导:
愈伤组织:枸杞无菌苗叶片在MS+0.1mg/L6BA+0.5mg/LNAA激素条件下诱导出来的无绿色芽点、质地紧密的愈伤组织为试验材料,在0.2%秋水仙素和2%DMSO混合液处理48h时诱导率高达66.7%,且嵌合体少、同质性高,是较好的枸杞多倍体诱导的方法。
激活种子:将枸杞种子经常规消毒后浸于无菌水中,待种子露白时用0.2%秋水仙素和2%DMSO混合液浸泡24h时诱导率最高达8%,但嵌合体现象严重,多倍体的特征不稳定,表现出不同程度的回复突变。
体细胞胚:枸杞无菌苗叶片在含0.2mg/L2,4-D的MS培养基上诱导出来的愈伤组织在相同培养基上继代培养4次后,转入不含激素的MS培养基上分化10天所得到的球形胚为试验材料,在20mg/L秋水仙素和1%DMSO混合液处理24h时诱变率6%、无嵌合体现象。本文首次报道,在体细胞胚的离体条件下,通过秋水仙素处理诱导获得了枸杞同源四倍体。
4.染色体计数鉴定表明,正常二倍体植株染色体数为24,所得到的四倍体植株染色体增加一倍,为48条染色体。
5.从形态学和细胞学方面对四倍体植株和二倍体植株进行了比较。结果显示,形态上四倍体比二倍体植株根系较少,但粗壮。四倍体植株与二倍体植株在气孔大小、频度具极显著差异。在40倍镜下,四倍体气孔长度31.9μm,二倍体植株气孔长度24.3μm;四倍体宽度29.0μm,二倍体宽度23.9μm。而在10倍镜视野内,气孔频度四倍体的21.6个,二倍体的是27个。四倍体气孔叶绿体数分布范围在24—50个,其中叶绿体数为38的占17%左右;二倍体气孔包围细胞叶绿体数分布范围在12—28个,其中叶绿体数为20的占25%。四倍体和二倍体气孔叶绿体数之比约等于2。
In recent years, the study of development of plant somatic embryo and technique of polyploid induction have become two important subjects in cytobiology and molecular biology. Many researches indicated that different valent metal ions had significantly different effect on adjusting somatic embryogenesis and had not same way of regulating for Lycium barbarum L.. However, the meaningful data was not sufficient to support it under same experimental condition. For polyploid induction, using somatic embryo to induce autotetraploid was also rarely reported.
In this study, Lycium barbarum L.(ninghong 1 hao) in Ningxia was used as materials for studying induction efficiency and relative growth rate of somatic embryo with three different explants ( root, stem and leaf) and different concentration of Ag~+、Ca~(2+) and Eu~(3+) as exogenous factor.
To induce the autotetraploid, somatic embryo, callus and seed were treated with different concentration of colchicines and DMSO mixture and was induced successfully into autotetraploidy plant from all of them. The inducing efficiency was compared for the three source of explant and some cytological and morphological analysis were also done to confirm the tetraploidy.
The objective of this study was to establish efficient somatic embryo induction system, optimize the technic of inducing plant autopolyploid and provide a method of cultivating homogeneity autotetraploid without chimera. The results are as follows:
1.When axenic Lycium barbarum L. had 3-4 leaves, the pieces of root , stem and leaf were induced on MS+2,4-D0.2mg/L. The result showed that the callus from leaf had highest of growth and the largest numbers of somatic embryo on MS medium without hormones. Leaf callus was more tight than that of stem and root. The relative growth rate of leaf callus reached 0.04g/(d·explant), while stem callus was (0.02g/(d·explant)) and root callus was (0.02g/(d·explant)); After cultured for 15 days on MS medium without hormones, the numbers of somatic embryo of a piece of callus was: 39.1 (root),42.8 (stem), 44.9 (leaf).
2.After 4 generation subculture on MS+2,4-D0.2mg/L, the callus induced from root, stem and leaf were transfered to MS medium with different concentration Ag~+、Ca~(2+) and Eu~(3+) for comparing the metal ion effect on embryogenesis. The result showed that low level of Ag~+、Ca~(2+) and Eu~(3+) raised the number of the embryo, but high level reduced it. The change tendency was same for three different explants.
When the concentration of Ag~+ was 1mg/L, it raised the number of somatic embryo as 2 times as that of control group. The promoting effect of Ca~(2+) and Eu~(3+) was not as good as Ag~+ when in their best level.
3.polyploid induction
From Callus, which was induced from leaves cultured on MS+ 6-BA0.1mg/L+ NAA0.1mg/L, the tight and colourless primordium cut from this callus was treated with0.2%colchicines and2%DMSO mixture for 48h and the 66.7% polyploid induction rate was get after cultured on the original medium, more than this, little chimera to be find.
From germinated seed, which was sterilized and germinated in sterile water, the rooting seed was treated with 0.2% colchicine and 2%DMSO mixture for 24h and 8% polyploid induction rate was get after cultured on the MS medium, but the polyploid was unstable because of chimerism and reversal mutation.
From somatic embryo, the globular embryoid induced from leaf by above mentioned method was treated with 20mg/L colchicine and 1%DMSO mixture medium for 24h, 6% polyploid induction rates was get without chimerism. This is the first report for inducing autotetraploid of Lycium barbarum L. from somatic embryo.
4.Chromosome number counting assay showed that the chromosome number of normal diploid is 24 and that of the polyploid is 48. This is confirmed the tetraploidy of the polyploid get in this experiment.
5.The morphological and cytological study indicated that the tetraploid grown slowly, the green color of its leaf was deep, its stem was stronger, its root was fewer but stronger, compared to diploid.
Comparing stomata of tetraploid and diploid also gave significant difference in number and size of pore. The data is as follows:
Under light microscope with 40×, for length of pore, tetraploid (31.9um). diploid.( 24.3um), for width, tetraploid (29.0um). diploid.(23.9um).
Under light microscope with 10×, for frequency ( number of pore in one field):tetraploid (21.6). diploid.(27). For amount of chloroplasts in one stomata , tetraploid ( 24~ 50) , 17% stomata was 38; diploid (12~ 48), 25% stomata was 20. So, the amount of chloroplast in defence cell of tetraploid is as about 2 times as that of diploid.
引文
[1] Steward F C,Mapes M O,Meats K.Growth and organized development of cultured cellⅡ.Organezation in cultures growth from freely suspended cells [J].America Journal of Botany,1958,45:705-708.
[2] 邢更生,邢更妹,崔凯荣等.枸杞胚性细胞分化的超微结构研究[J].电子显微学报,1999,18(4):395-399.
[3] 朱微.植物组织培养中的胚状体[J].遗传学报,1978(5):79-88.
[4] 周俊彦.植物体细胞胚在组织培养中产生的胚状体Ⅰ.植物体细胞胚的胚状体发生[J].植物生理与分子生物学学报,1981,(7):389-395.
[5] ulecke W.Cell and Tissue Culture in Forestry VolⅡ[M].Dordrecht,Boston,Lancaster:Martinus Nijhoff Publishers,1987.
[6] Yantcheva A,Vlahova M,Antanassov A.Direct somatic embryogenesis and Plant regeneration of carnation (Dianthus Caryophyllus L.) [J].Plant Cell Rep,1998,18:148-153.
[7] Chen JT,Chang W C.Direct somatic embryogensis on leaf explants of on cidum Gower Ransey and subsequent plant regeneration [J].Plant Cell Rep,1999,19:143-149.
[8] Sarsan V,Ramsay M M,Reberts A V.Intro germination and induction of direct somatic embryogensis in“Bottle plam”[Hyophorbe lagenicauli(L.Bailey)H.E.Moore],acritically endangenered Mauritian plam [J].Plant Cell Rep,2002,20:1107-1111.
[9] Lin H S,Toom C,Raemakers K J J M et al.Development of a plant regeneration system based on friable embryogenic callus in the ornamental Alstroemeria [J].Plant Cell Rep,2000,19:529-534.
[10] 周俊彦.植物细细胞在组织培养中产生胚状体[J].植物生理学报,1981,7(4):329-397.
[11] Plata E,Vieitez Ana M.In vitro regeneration of Camellia reticulata by somatic embryogensis [J].Horti.Sci,1990,65:707-714.
[12] 崔凯荣,邢更生,周攻克等.体细胞胚发生的生化基础[J].生命科学,2001,13(1)28-33.
[13] 李杉,邢更妹,崔凯荣等.植物体细胞胚发生中 ATP 酶活性时空分布动态与内源激素的变化[J].植物学通报,2001,18(3):308-317.
[14] 高述民,陆帼一,杜慧芳.大蒜体细胞胚发育分化中特异蛋白和某些生理生化变化[J].植 物生理学通讯,2001,37(3):207-210.
[15] 胡忠,刘天磊,江晓雯等.宁夏枸杞愈伤组织器官发生和体细胞胚发生过程中过氧化物酶和酸性磷酸酶同工酶的研究[J].兰州大学学报,2000,36(3):147-153.
[16] Jain R K,Maherckandani N,Chow dhury V K et al.Radiation-induced organogenesis and isoenzyme patterns in long-term callus cultures of Datura innoxia [J].Ann Bot,1990,65:659-663.
[17] 崔凯荣,王晓哲,陈雄等.小麦体细胞胚发生中DNA,RNA和蛋白质合成动态[J].核农学报,1997,11(4):283-386.
[18] Kopertekh L G,Butenko R G.Naturally occurring phytohormones in wheat explants as ralated to wheat morphogenesis in vitro [J].Plant Physiol,1995,42:488-491.
[19] Jimenez V M.Regulation of in vitro somatie embryogenesis with emphasis on the role of endogenous hormones [J].Rev Bras Fisiol Veg,2001,13:196-223.
[20] Cooke T J,Racusen R H,Cohen JD.The role of auxin in Plant embryogenesis [J].Plant Cell,1993,(5):1494-1495.
[21] Fischer C,Neuhaus G.Influence of auxin on the establishment of bilateral symmetry inmonocots [J].Plant Physiol,1996,9:659-69.
[22] Pastermak T P,Prinsen E,Ayaydin F et al.The role of auxin,PH and stress in the activation of embryogenic cell division in leaf protoplast-derived cells of alfalfa [J].Plant Physiol,2002,129:1807-1819.
[23] Charriere F,Sotta B,and Miginiac E et al.Induction of adventitious shoots or somatic embryos on in vitro cultured zygotic embryos of Helianthus annuus:variation of endogenous hormone levels [J].Plant Physiol,1999,37:751-757.
[24] Kiyosue T,Satoh S,Kamada H et al.Somatic embryogenesis in higher Plants [J].Plant Res, 1993,3:75-82.
[25] Gazzarrini S,McCourt P.Cross-talk in plant hormone signaling:what arabidopsis mutants are telling us [J].Ann.Bot,2003,91:605-612.
[26] Jimenez V M,Bangerth F.In vitro culture and endogenous hormone levels in immature zygotic embryos,endosperm and callus cultures of normal and high-lysine barley genotypes [J].Appi.Bot,2001,75:1-7.
[27] Rajasekaran K,Hein M B,Vasil I K.Endogenous abscisic acid and indole-3-acetic acid and somatic embryogenesis in cultured leaf explants of pennisetum purpureum schum [J].Plant Physiol,1987,84:47-51.
[28] Ivanova A,Velcheva M,Deneltev P et al.Endogenous hormone levels during direct somatic embryogenesis in Medicago falcata [J].Plant Physiol,1994,92:85-89.
[29] Guiderdoni E,Me,rot B,Eksomtramage T et al.Somatic embryogenesis in sugareane(Saecharum species).In:Bajaj Y.P.S.(ed.),Somatic embryogenesis and synthetic Seed [J].Biotechnology in Agriculture and Forestry,1995,31:92-113.
[30] Michalezuk L,Drulart P.Indole-3-acetic acid metabolism in hormone-autotrophic,embryogenic callus of Inmil-cherry rootstoek and inhormone-dependent,nonembryogenic calli of Prunus incise serrula and Prunus domestica [J].Plant Physiol,1999,107:426-432.
[31] Fcher A,Pasernak T P,Dudits D.Transition of somatie Plant cells to an embryogenic state [J].Plant Cell Tiss,org.Cult,2003,74:201-228.
[32] Kiyosue T,Satoh S,Kamada H et al.Somatic embryogenesis in higher Plants [J].Plant Res, 1993,3:75-82.
[33] Hideki Nakagawa,Takeshi S,Naoki Y et al.Effect of sugars and abscisic acid on somatic Embryogensis from melon (cucumis melo L.) expanded cotyledon [J].Scientia Horticulturae,2001,90:85-92.
[34] Etienne H,Sotta B,Montoro P et al.Relation between exogenous growth regulators and endogenous indole-3-acetic acid and abscisic acid in the expression of somatic embryogenesis in Hevea brasiliensis (MuⅡ.Arg.) [J].Plant Sci,1993,88:91-96.
[35] Pintos B,Marti,n J P,Centeno M L et al.Endogenous cytokinin levels in embryogensis and non-embryogensis calli of Medicago arborea L.[J].Plant Sci,2002,163:955-960.
[36] Gaj M D.Factors influencing somatic embryoegnsis induction and plant regeneration with particular reference to Arabidopsis thaliana L.Heynh [J].Plant Growth Regulat,2004,43:27-47.
[37] Lakshmanan P,Taji A.Somatic embryogenesis in leguminous Plants [J].Plant Biol,2000,2: 136-148.
[38] Panaia M,Senaratna T,Dixon K W et al.The role of cytokinins and thidiazuron in the stimulation of somatic embryogensis in key members of the restionaceae [J].Aust Bot,2004,52:257-267
[39] Hernandez I,Celestino C,Toribio M.Vegetative propagation of quereus suber L.by somatic embryogenesis [1] Factors affecting the induction in leaves from mature corkoak trees [J].Plant Cell Rep,2003,21:759-764.
[40] Bell L M,Trigiano R N,Conger B V.Relationship of abscisic acid to somatic embryogenesis in Dactylis glomerata environmental and experimental botany [J].Plant Cell Rep,1993,33,(4): 495-499.
[41] Seabrook J E K,Douglass L K.Somatic embryogenesis on various Potato tissues from a range of genotypes and ploidy levels [J].Plant Cell Rep,2001,20:175-182.
[42] Parrott W A,Merkle S A,Williams E G.Somatic embryogenesis:Potential for use in propagation and gene transfer systems ln:Cab Int.Murray DR(Ed) Advaneed Methods in Plant Breeding and Biotechnology Y K:Wallingford [J].Plant sci,2000,158-200.
[43] Chengalryan K,Mhaske V B,Hazra S.Genotypic control of peanuts somatic embryogenesis [J].Plant Cell Rep,1998,17:522-525.
[44] He D G et al.Transformation of wheat (Triticum aestivum L.) through electroporation of protoplasts [J].Plant sci,1986,45:119-124.
[45] Kiyosue T,Takano K,Kamada H et al.Induction of somatic embryogensis in carrot by heavy metalions [J].Can J Bot,1990,68:2301-2303.
[46] He D G,Yang Y M,Scott K J.Zinc deficiency and the formation of white structures in immature entry cultures of wheat (Triticium aesticum L.) [J].Plant Cell Tissue and Organ Culture,1991,24:9-12.
[47] Rougtan J P,Latche A,Fallot J.Stimulation of Daucus carota somatic embryogensis by inhibitors of ethylene synthesis:cobalt and nickel [J].Plant cell Rep,1989,8:182-185.
[48] 杨金岭,桂耀林,杨映根.白杄体细胞胚发生及其植株再生[J].植物学报,1997,39(4):315-321.
[49] 周晓波,魏幼璋. 稀土离子与 Ca2+在生物体内的相互作用机制及应用[J].生命科学,1999,11(增刊):70-74.
[50] 申治国,雷衡毅,项辉等.金属离子对钙调素生物功能影响的研究进展[J].生命科学,1999,11(增刊):74-77.
[51] 向太和,钟华鑫,梁海曼.水稻胚性悬浮系建立过程中的生理生化变化.Ⅱ.氨基酸、多胺及内源激素的变化[J].作物学报,1995,21(2):223-229.
[52] 崔凯荣,戴若兰.植物体细胞胚发生的分子生物学[M].科学出版社,2000,1-2.
[53] 李杉,秦芝,王亚馥.枸杞体细胞胚发生中对痕量金属离子吸收动态研究[J].植物学报,2001,43(10):1031-1035.
[54] Li S,Qin Z,Dai R L.Preparation of multitracer from silver target irradiated with 80 Mevnuceon Ne-20ions [J].Journal of Radioanalytical and Nuclear Chemistry,2001,247(3):545-548.
[55] Li S,Qin Z,Wang Y F.Uptake rate of multitration of tracer element by Lycium barbarum L.somatic embryogensis [J].Journal of Radionalytical and Nuclear Chemistry,2001,250(3): 593-597.
[56] Li S,Qin Z,Wang Y F.Effect of Ag+ concentration on the uptake rate of tracer elements duringthe somatic embryogenesis of lycium barbarum L.[J].Journal of Radioanalytical and Nuclear Chemistry,2001,250(3):599-602.
[57] Schmidt E D L,Guzzo F,Toonen M A J et al.A Leucine-rich containing receptor-like kinase marks somatic plant cells competent to from embryos [J].Development,1997,124:2049-2062.
[58] Somleva M N,Schmidt E D L,Vries S C.Embryogenic cells in Dacylis glomerata L.(Poaceae) explants identified by cell tracking and SERK expression [J].Plant Cell Rep,2000,19:718-726.
[59] Hecht V,Vielle Calzada J P,Vriesetal.The Arabdopsis somatic embryogenesis receptor kinase l gene is expressed in developing ovules and embryos and enhances embryogenic competence in culture [J].Plant Physiol,2001,127:803-816.
[60] Baudino S,Hansen S,Brettschneider R et al.Molecular characterization of two novel maize LRR receptor-like kinase,which belong to the SERK gene family [J].Planta,2001,213:1-10.
[61] Nolan K E,Irwanto R R,Rose R J.Auxin up-regulates Mt SERK1 expression in both Medicago truncatula root-froming and embryogenesis culture [J].Plant Physiol,2003,133:218-230.
[62] Santos M O,Romano E,Yotoko K S C et al.Characterization of the cacao somatic embryogensis receptor-like kinase (SERK) gene expressed during somatic embryogensis [J].Plant Science,2005,168:723-729.
[63] Chandel G,Katiyar S K.Organogenesis and somatic embryogensis in tomoto (Lycopersicon esculantum Mill) [J].Adv Plant Sci,2000,13:11-17.
[64] 陈佩度.作物育种生物技术[M].北京:中国农业出版社,2001,5.
[65] 王兴智.六倍体小黑麦与普通小麦杂种 F1 花粉植株的细胞遗传学研究[J].遗传学报,1984,11(1):33-38.
[66] 申馥玉.花生种间三倍体杂种染色体加倍技术的研究[J].中国农业科学,1984,4:21-25.
[67] 李文泽.植物生物技术与作物改良[M].中国科学技术出版社,1995,9.
[68] Zhang H M,Xu T F,Guo M L et a1.Polyploid breeding of medicinal plant [J].Chinese traditional and Herbal Drugs,2002,33(7):1-3.
[69] 武振华,牛炳韬,王新宇.药用植物染色体加倍的研究进展[J].西北植物学报,2005,25,(12):2569-2574.
[70] 乔传卓,崔熙.药用植物多倍体的应用[J].中药材科技,1981,4(4):40.
[71] 陈柏君,高山林,卞云云.黄答组织培养同源四倍体的诱导[J].植物资源与环境学报,2000, 9(l):9-11.
[72] 彭锐,张明.多倍体及其在中药材生产上的应用[J].重庆中草药研究,2000,(41):14.
[73] 朱英,来平凡.石菖蒲的研究近况[J].浙江中医学院学报,2001,25(3):80.
[74] 吕世民,梁可钧,葛传吉等.怀牛膝多倍体育种的研究[J].中药通报,1988,13(7):395.
[75] 程心昊,高山林,卞云云.白术同源四倍体的诱导和鉴定及其与二倍体过氧化物酶的比较[J].植物资源与环境学报,2003,12(1):16.
[76] 彭菲,张胜,刘塔斯等.四倍体白芷药材中欧前胡素的含量测定[J].中国中药杂志,2002,27(6):426.
[77] Soltis D E,Soltis P S.Polyploidy recurrent formation and genomeevolution [J].Trends Ecol Evol,1999,9:348.
[78] 乔传卓,吴美枢,戴富宝等.菘蓝多倍体育种的研究[J].植物学报,1989,31(9):678.
[79] 艾建国,高山林.丹参同源四倍体的诱导、鉴定及有效成分的含量测定[J].药用生物技术,2003,10(6):372-376.
[80] Gao S L,Chen B J,Zhu D N.In vitro production and identification of autotetraploids of scutellaria baicalensis [J].Plant Cell Tissue Organ Culture,2002,70:289–293.
[81] Petersen K K,Hagberg P,Kristiansen K.Colchicine and oryzalin mediated chromosome doubling in different genotypes of Miscanthus sinensis [J].Plant Cell Tissue Organ Culture,2003,73: 137-146.
[82] Rose J B,Kubba J,Tobutt K R.Chromosome doubling in sterile Syringa vulgaris × S.pinnatifolia hybrids by in vitro culture of nodal explants [J].Plant Cell Tissue Organ Culture,2000,63:127-132.
[83] Roy A T,Leggett G,Koutoulis A.In vitro tetraploid induction and generation of tetraploids from mixoploidsin hop (Humulus lupulus L.) [J].Plant Cell Rep,2001,20(6):489-495.
[84] Wu J H,Mooney P.Autotetraploid tangor plant regeneration from in vitro Citrus somatic embryogenic callus treated with colchicines [J].Plant Cell Tissue Organ Culture,2002,70:99-104.
[85] 李贤,钟仲贤.植物离体组织细胞染色体加倍技术在育种中的应用[J].上海农业学报,1998,14(3):99-102.
[86] 吴清,向素琼,严勇等.金荞麦的离体繁殖及同源四倍体的诱导[J].西南农业大学学报,2002,3(2):108-110.
[87] 常月梅.果树多倍体鉴定进展[J].山西林业科技,2000,3(l):l.
[88] 王丽艳,梁国鲁.植物多倍体的形成途径及鉴定方法[J].北方园艺,2004,(l):61.
[89] 郭启高,宋明,梁国鲁.植物多倍体诱导育种研究进展[J].生物学通报,2000,35(2):8.
[90] Sari N,Abak K,Pitrat M.Comparison of ploidylevel screening methods in watermelon: Citrullus lanatus (Thunb.) [J].Scientia Horticulturae,1999,82:265-277.
[91] Tambong J T,Sapra V T,Garton S.In vitro induction of tetraploids in colchicines-treated cocoyam plantlets [J].Euphytica,1998,104:191-197.
[92] Compton M,Barnett N,Gray D.Use of fluoresceindiacetate (FDA) to determine ploidy of invitro watermelon shoots [J].Plant Cell,Tissue and Organ Culture,1999,58:199-203.
[93] 刘文革,阉志红,饶小利.不同倍性西瓜的叶表皮微形态特征比较[J].果树学报,2005,22:31-34.
[94] Cohen D,Yao J.In vitro chromosome doubling of nine Zantedeschia cultivars [J].Plant Cell, Tissue and Organ Culture,1996,47:43-49.
[95] 杨瑞芳,郑思乡,郭清泉等.莲多倍体研究、莲花粉母细胞减数分裂行为观察[J].湖南农业大学学报,1998,24(2):95.
[96] 李愈学,张赞平.作物染色体及研究技术汇[M].北京:中国农业出版社,1990,10.
[97] 李步勋,陶抵辉,阮万辉.西瓜离体组织细胞染色体加倍技术的研究和应用[J].陕西农业科学,1999,(3):21.
[98] 秦盘山,王莉,陈素萍等.枸杞同源四倍侔的诱导与应用研究[A].自寿宁.宁夏枸杞研究[C].银川:宁夏人民出版社,1988:85-88.
[99] 艾先元,石巍峨,荆雅琴.枸杞茎尖培育四倍体苗初报[J].宁夏农林科技,I991.5-32.
[100] 王莉,陈紊萍,秦金山等.枸杞胚乳植株诱导和它的倍性水平[J].檀精学报,1985,12(6):440-444.
[101] 王莉,陈紊萍,秦金山等.枸杞胚乳植株的诱导及染色体倍性观察[J].中国科学(B辑),1986,690-606.
[102] 厦飒荣,桂耀林,棣廷玉.枸杞胚乳植株的诱导[J].植物学报,1885.27(1):106-109.
[103] 硕淑荣,桂耀韩.撩廷玉.枸杞胚乳植株的诱导及染色体倍性观察[A].自寿宁.宁夏枸杞研究[C].银川:宁夏人民出版社,1998:89-92.
[104] 马爱如,牛一恕.诱导宁夏拘杞多倍体研究初报[J].北农业科学,1987,6:26-27.
[105] 马爱如,牛一恕.三倍体宁夏枸杞研究初报[J].湖北农业科学,1988,9:31-32.
[106] 钟牲元,王燕,王锦秀.无耔拘杞选育初报[J].宁夏农林科技,1993,3:15-17.
[107] 安巍,李立精,焦忘宁等.三倍体无耔枸记新品种的选育研究[J].宁夏农林科技,1989,2:21-24.
[108] 张新宁,沈效东,王锦绣.枸杞四倍体同二倍体杂交败育的形态分析及解决方法[J].宁夏农林科技,1992(1):30-32.
[109] 厦飒荣,桂耀林,棣廷玉.枸杞胚乳植株的诱导[J].植物学报,1885,27(1):106-109.
[110] 陈紊萍,王莉,束秀清等.枸杞胚乳植株二代的形态及细胞学观察[A].自寿宁.宁夏枸杞研究[C].银川:宁夏人品出版社,1998,104-106.
[111] 王慧中,黄发蚰,李安生等.枸杞转基因植株的再生[A]白寿宁.宁夏枸杞研究[C]银川:宁夏人民出版社,1998:114-146.
[112] 王慧中,杜立群,黄发灿等.根瘤农杆菌介导的枸杞转化及转化植株的获得[J].中国科学(B辑),1993,23(4):391-395.
[113] Wu J H,Auline Moone.Totetraploid tangor plant regeneration from in vitro citrus somatic embryogensis callus treated with colchicines[J].Plant Cell Tissue and Organ Culture,2002,70:99-104.
[114] Yang X M,Cao Z Y,An L Z et al.In vitro tetraploid induction via colchicine treatment from diploid somatic embryos in grapevine ( Vitis vinifera L.) [J].Euphytica,2006,152:217–224.
[115] Murashige T,Skoog F.A revised medium for rapid growth and bioassays with tobacco tissue cultures [J].Plant Physiol,1962,15,473-497.
[116] Singer S R.Analysis growth in cell culture:calculating growth rates [J].Can J Bot,1986,64: 33-237.
[117] Fuentes S R L,Calheiros M B P,Manetti-Filho J et al.The effects of silver nitrate and different carbohydrate sources on somatic embryogenesis in Coffea canephora [J].Plant Cell Tiss Org Cult, 2000 (60):5-13.
[118] 崔凯荣,王晓哲,王亚馥.植物体细胞胚发生研究的一些现状[J].植物学通报,1993,10(3):14-20.
[119] Kong L,Yeung E C.Effects of ethylene and ethylene inhibitors on white spruce somatic embryo maturation [J].Plant Sci,1994(104):71-80.
[120] 杨汉民,高清祥,张爱国等.枸杞体细胞胚的诱导与形态发生[J].兰州大学学报,1992,28(1):87-95.
[121] Kadota M,Niimi Y.In vitro induction of tetraploid plants from a diploid Japanese pear cultivar (Pyrus pyrifolia N.cv.Hosui) [J].Plant Cell Rep,2002,21:282-286.
[122] Thao N T P,Ureshino K,Miyajima I et al.Induction of tetraploid in ornamental Alocasia through colchicine and oryzalin treatments [J].Plant Cell Tissue Organ Culture,2003,72:19-25.
[123] Michael M Blanke,Monika H?fer,Richard J.Pring Stomata and Structure of Tetraploid AppleLeaves cultured in Vitro Annals of Botany [J].Scientia Horticulturae,1994,73 (6):651-654.
[124] Chen L L,Gao S L.In vitro tetraploid induction and generation of tetraploids from mixoploids in Astragalus membranaceus [J].Plant Cell Rep,2001,112(3):339-344.
[125] 匡全,梁国鲁,郭启高等.秋水仙素诱导牛蒡多倍体[J].植物生理学通讯,2004,40(2): 157.
[126] Ollitroult Sammarcell.Ploidy levels in Kiwifrtui Plants determ inated by flow cytometer [J].Hort.Sci,1994,57(4):303-314.
[127] Costich D E Flow eytometer fou rapid determination of ploidy level in Vaceiniun [J].Theoretieal and Applied Geneties,1995,30(6):1276-1278.
[128] Leblanc O,Duenas M,Hemandez M et al.Chromosome doubling in Tripsacum:the production of artificial,sexual tetraploid plants [J].Plant-breeding,1995,114(3):226-230.
[2] 邢更生,邢更妹,崔凯荣等.枸杞胚性细胞分化的超微结构研究[J].电子显微学报,1999,18(4):395-399.
[3] 朱微.植物组织培养中的胚状体[J].遗传学报,1978(5):79-88.
[4] 周俊彦.植物体细胞胚在组织培养中产生的胚状体Ⅰ.植物体细胞胚的胚状体发生[J].植物生理与分子生物学学报,1981,(7):389-395.
[5] ulecke W.Cell and Tissue Culture in Forestry VolⅡ[M].Dordrecht,Boston,Lancaster:Martinus Nijhoff Publishers,1987.
[6] Yantcheva A,Vlahova M,Antanassov A.Direct somatic embryogenesis and Plant regeneration of carnation (Dianthus Caryophyllus L.) [J].Plant Cell Rep,1998,18:148-153.
[7] Chen JT,Chang W C.Direct somatic embryogensis on leaf explants of on cidum Gower Ransey and subsequent plant regeneration [J].Plant Cell Rep,1999,19:143-149.
[8] Sarsan V,Ramsay M M,Reberts A V.Intro germination and induction of direct somatic embryogensis in“Bottle plam”[Hyophorbe lagenicauli(L.Bailey)H.E.Moore],acritically endangenered Mauritian plam [J].Plant Cell Rep,2002,20:1107-1111.
[9] Lin H S,Toom C,Raemakers K J J M et al.Development of a plant regeneration system based on friable embryogenic callus in the ornamental Alstroemeria [J].Plant Cell Rep,2000,19:529-534.
[10] 周俊彦.植物细细胞在组织培养中产生胚状体[J].植物生理学报,1981,7(4):329-397.
[11] Plata E,Vieitez Ana M.In vitro regeneration of Camellia reticulata by somatic embryogensis [J].Horti.Sci,1990,65:707-714.
[12] 崔凯荣,邢更生,周攻克等.体细胞胚发生的生化基础[J].生命科学,2001,13(1)28-33.
[13] 李杉,邢更妹,崔凯荣等.植物体细胞胚发生中 ATP 酶活性时空分布动态与内源激素的变化[J].植物学通报,2001,18(3):308-317.
[14] 高述民,陆帼一,杜慧芳.大蒜体细胞胚发育分化中特异蛋白和某些生理生化变化[J].植 物生理学通讯,2001,37(3):207-210.
[15] 胡忠,刘天磊,江晓雯等.宁夏枸杞愈伤组织器官发生和体细胞胚发生过程中过氧化物酶和酸性磷酸酶同工酶的研究[J].兰州大学学报,2000,36(3):147-153.
[16] Jain R K,Maherckandani N,Chow dhury V K et al.Radiation-induced organogenesis and isoenzyme patterns in long-term callus cultures of Datura innoxia [J].Ann Bot,1990,65:659-663.
[17] 崔凯荣,王晓哲,陈雄等.小麦体细胞胚发生中DNA,RNA和蛋白质合成动态[J].核农学报,1997,11(4):283-386.
[18] Kopertekh L G,Butenko R G.Naturally occurring phytohormones in wheat explants as ralated to wheat morphogenesis in vitro [J].Plant Physiol,1995,42:488-491.
[19] Jimenez V M.Regulation of in vitro somatie embryogenesis with emphasis on the role of endogenous hormones [J].Rev Bras Fisiol Veg,2001,13:196-223.
[20] Cooke T J,Racusen R H,Cohen JD.The role of auxin in Plant embryogenesis [J].Plant Cell,1993,(5):1494-1495.
[21] Fischer C,Neuhaus G.Influence of auxin on the establishment of bilateral symmetry inmonocots [J].Plant Physiol,1996,9:659-69.
[22] Pastermak T P,Prinsen E,Ayaydin F et al.The role of auxin,PH and stress in the activation of embryogenic cell division in leaf protoplast-derived cells of alfalfa [J].Plant Physiol,2002,129:1807-1819.
[23] Charriere F,Sotta B,and Miginiac E et al.Induction of adventitious shoots or somatic embryos on in vitro cultured zygotic embryos of Helianthus annuus:variation of endogenous hormone levels [J].Plant Physiol,1999,37:751-757.
[24] Kiyosue T,Satoh S,Kamada H et al.Somatic embryogenesis in higher Plants [J].Plant Res, 1993,3:75-82.
[25] Gazzarrini S,McCourt P.Cross-talk in plant hormone signaling:what arabidopsis mutants are telling us [J].Ann.Bot,2003,91:605-612.
[26] Jimenez V M,Bangerth F.In vitro culture and endogenous hormone levels in immature zygotic embryos,endosperm and callus cultures of normal and high-lysine barley genotypes [J].Appi.Bot,2001,75:1-7.
[27] Rajasekaran K,Hein M B,Vasil I K.Endogenous abscisic acid and indole-3-acetic acid and somatic embryogenesis in cultured leaf explants of pennisetum purpureum schum [J].Plant Physiol,1987,84:47-51.
[28] Ivanova A,Velcheva M,Deneltev P et al.Endogenous hormone levels during direct somatic embryogenesis in Medicago falcata [J].Plant Physiol,1994,92:85-89.
[29] Guiderdoni E,Me,rot B,Eksomtramage T et al.Somatic embryogenesis in sugareane(Saecharum species).In:Bajaj Y.P.S.(ed.),Somatic embryogenesis and synthetic Seed [J].Biotechnology in Agriculture and Forestry,1995,31:92-113.
[30] Michalezuk L,Drulart P.Indole-3-acetic acid metabolism in hormone-autotrophic,embryogenic callus of Inmil-cherry rootstoek and inhormone-dependent,nonembryogenic calli of Prunus incise serrula and Prunus domestica [J].Plant Physiol,1999,107:426-432.
[31] Fcher A,Pasernak T P,Dudits D.Transition of somatie Plant cells to an embryogenic state [J].Plant Cell Tiss,org.Cult,2003,74:201-228.
[32] Kiyosue T,Satoh S,Kamada H et al.Somatic embryogenesis in higher Plants [J].Plant Res, 1993,3:75-82.
[33] Hideki Nakagawa,Takeshi S,Naoki Y et al.Effect of sugars and abscisic acid on somatic Embryogensis from melon (cucumis melo L.) expanded cotyledon [J].Scientia Horticulturae,2001,90:85-92.
[34] Etienne H,Sotta B,Montoro P et al.Relation between exogenous growth regulators and endogenous indole-3-acetic acid and abscisic acid in the expression of somatic embryogenesis in Hevea brasiliensis (MuⅡ.Arg.) [J].Plant Sci,1993,88:91-96.
[35] Pintos B,Marti,n J P,Centeno M L et al.Endogenous cytokinin levels in embryogensis and non-embryogensis calli of Medicago arborea L.[J].Plant Sci,2002,163:955-960.
[36] Gaj M D.Factors influencing somatic embryoegnsis induction and plant regeneration with particular reference to Arabidopsis thaliana L.Heynh [J].Plant Growth Regulat,2004,43:27-47.
[37] Lakshmanan P,Taji A.Somatic embryogenesis in leguminous Plants [J].Plant Biol,2000,2: 136-148.
[38] Panaia M,Senaratna T,Dixon K W et al.The role of cytokinins and thidiazuron in the stimulation of somatic embryogensis in key members of the restionaceae [J].Aust Bot,2004,52:257-267
[39] Hernandez I,Celestino C,Toribio M.Vegetative propagation of quereus suber L.by somatic embryogenesis [1] Factors affecting the induction in leaves from mature corkoak trees [J].Plant Cell Rep,2003,21:759-764.
[40] Bell L M,Trigiano R N,Conger B V.Relationship of abscisic acid to somatic embryogenesis in Dactylis glomerata environmental and experimental botany [J].Plant Cell Rep,1993,33,(4): 495-499.
[41] Seabrook J E K,Douglass L K.Somatic embryogenesis on various Potato tissues from a range of genotypes and ploidy levels [J].Plant Cell Rep,2001,20:175-182.
[42] Parrott W A,Merkle S A,Williams E G.Somatic embryogenesis:Potential for use in propagation and gene transfer systems ln:Cab Int.Murray DR(Ed) Advaneed Methods in Plant Breeding and Biotechnology Y K:Wallingford [J].Plant sci,2000,158-200.
[43] Chengalryan K,Mhaske V B,Hazra S.Genotypic control of peanuts somatic embryogenesis [J].Plant Cell Rep,1998,17:522-525.
[44] He D G et al.Transformation of wheat (Triticum aestivum L.) through electroporation of protoplasts [J].Plant sci,1986,45:119-124.
[45] Kiyosue T,Takano K,Kamada H et al.Induction of somatic embryogensis in carrot by heavy metalions [J].Can J Bot,1990,68:2301-2303.
[46] He D G,Yang Y M,Scott K J.Zinc deficiency and the formation of white structures in immature entry cultures of wheat (Triticium aesticum L.) [J].Plant Cell Tissue and Organ Culture,1991,24:9-12.
[47] Rougtan J P,Latche A,Fallot J.Stimulation of Daucus carota somatic embryogensis by inhibitors of ethylene synthesis:cobalt and nickel [J].Plant cell Rep,1989,8:182-185.
[48] 杨金岭,桂耀林,杨映根.白杄体细胞胚发生及其植株再生[J].植物学报,1997,39(4):315-321.
[49] 周晓波,魏幼璋. 稀土离子与 Ca2+在生物体内的相互作用机制及应用[J].生命科学,1999,11(增刊):70-74.
[50] 申治国,雷衡毅,项辉等.金属离子对钙调素生物功能影响的研究进展[J].生命科学,1999,11(增刊):74-77.
[51] 向太和,钟华鑫,梁海曼.水稻胚性悬浮系建立过程中的生理生化变化.Ⅱ.氨基酸、多胺及内源激素的变化[J].作物学报,1995,21(2):223-229.
[52] 崔凯荣,戴若兰.植物体细胞胚发生的分子生物学[M].科学出版社,2000,1-2.
[53] 李杉,秦芝,王亚馥.枸杞体细胞胚发生中对痕量金属离子吸收动态研究[J].植物学报,2001,43(10):1031-1035.
[54] Li S,Qin Z,Dai R L.Preparation of multitracer from silver target irradiated with 80 Mevnuceon Ne-20ions [J].Journal of Radioanalytical and Nuclear Chemistry,2001,247(3):545-548.
[55] Li S,Qin Z,Wang Y F.Uptake rate of multitration of tracer element by Lycium barbarum L.somatic embryogensis [J].Journal of Radionalytical and Nuclear Chemistry,2001,250(3): 593-597.
[56] Li S,Qin Z,Wang Y F.Effect of Ag+ concentration on the uptake rate of tracer elements duringthe somatic embryogenesis of lycium barbarum L.[J].Journal of Radioanalytical and Nuclear Chemistry,2001,250(3):599-602.
[57] Schmidt E D L,Guzzo F,Toonen M A J et al.A Leucine-rich containing receptor-like kinase marks somatic plant cells competent to from embryos [J].Development,1997,124:2049-2062.
[58] Somleva M N,Schmidt E D L,Vries S C.Embryogenic cells in Dacylis glomerata L.(Poaceae) explants identified by cell tracking and SERK expression [J].Plant Cell Rep,2000,19:718-726.
[59] Hecht V,Vielle Calzada J P,Vriesetal.The Arabdopsis somatic embryogenesis receptor kinase l gene is expressed in developing ovules and embryos and enhances embryogenic competence in culture [J].Plant Physiol,2001,127:803-816.
[60] Baudino S,Hansen S,Brettschneider R et al.Molecular characterization of two novel maize LRR receptor-like kinase,which belong to the SERK gene family [J].Planta,2001,213:1-10.
[61] Nolan K E,Irwanto R R,Rose R J.Auxin up-regulates Mt SERK1 expression in both Medicago truncatula root-froming and embryogenesis culture [J].Plant Physiol,2003,133:218-230.
[62] Santos M O,Romano E,Yotoko K S C et al.Characterization of the cacao somatic embryogensis receptor-like kinase (SERK) gene expressed during somatic embryogensis [J].Plant Science,2005,168:723-729.
[63] Chandel G,Katiyar S K.Organogenesis and somatic embryogensis in tomoto (Lycopersicon esculantum Mill) [J].Adv Plant Sci,2000,13:11-17.
[64] 陈佩度.作物育种生物技术[M].北京:中国农业出版社,2001,5.
[65] 王兴智.六倍体小黑麦与普通小麦杂种 F1 花粉植株的细胞遗传学研究[J].遗传学报,1984,11(1):33-38.
[66] 申馥玉.花生种间三倍体杂种染色体加倍技术的研究[J].中国农业科学,1984,4:21-25.
[67] 李文泽.植物生物技术与作物改良[M].中国科学技术出版社,1995,9.
[68] Zhang H M,Xu T F,Guo M L et a1.Polyploid breeding of medicinal plant [J].Chinese traditional and Herbal Drugs,2002,33(7):1-3.
[69] 武振华,牛炳韬,王新宇.药用植物染色体加倍的研究进展[J].西北植物学报,2005,25,(12):2569-2574.
[70] 乔传卓,崔熙.药用植物多倍体的应用[J].中药材科技,1981,4(4):40.
[71] 陈柏君,高山林,卞云云.黄答组织培养同源四倍体的诱导[J].植物资源与环境学报,2000, 9(l):9-11.
[72] 彭锐,张明.多倍体及其在中药材生产上的应用[J].重庆中草药研究,2000,(41):14.
[73] 朱英,来平凡.石菖蒲的研究近况[J].浙江中医学院学报,2001,25(3):80.
[74] 吕世民,梁可钧,葛传吉等.怀牛膝多倍体育种的研究[J].中药通报,1988,13(7):395.
[75] 程心昊,高山林,卞云云.白术同源四倍体的诱导和鉴定及其与二倍体过氧化物酶的比较[J].植物资源与环境学报,2003,12(1):16.
[76] 彭菲,张胜,刘塔斯等.四倍体白芷药材中欧前胡素的含量测定[J].中国中药杂志,2002,27(6):426.
[77] Soltis D E,Soltis P S.Polyploidy recurrent formation and genomeevolution [J].Trends Ecol Evol,1999,9:348.
[78] 乔传卓,吴美枢,戴富宝等.菘蓝多倍体育种的研究[J].植物学报,1989,31(9):678.
[79] 艾建国,高山林.丹参同源四倍体的诱导、鉴定及有效成分的含量测定[J].药用生物技术,2003,10(6):372-376.
[80] Gao S L,Chen B J,Zhu D N.In vitro production and identification of autotetraploids of scutellaria baicalensis [J].Plant Cell Tissue Organ Culture,2002,70:289–293.
[81] Petersen K K,Hagberg P,Kristiansen K.Colchicine and oryzalin mediated chromosome doubling in different genotypes of Miscanthus sinensis [J].Plant Cell Tissue Organ Culture,2003,73: 137-146.
[82] Rose J B,Kubba J,Tobutt K R.Chromosome doubling in sterile Syringa vulgaris × S.pinnatifolia hybrids by in vitro culture of nodal explants [J].Plant Cell Tissue Organ Culture,2000,63:127-132.
[83] Roy A T,Leggett G,Koutoulis A.In vitro tetraploid induction and generation of tetraploids from mixoploidsin hop (Humulus lupulus L.) [J].Plant Cell Rep,2001,20(6):489-495.
[84] Wu J H,Mooney P.Autotetraploid tangor plant regeneration from in vitro Citrus somatic embryogenic callus treated with colchicines [J].Plant Cell Tissue Organ Culture,2002,70:99-104.
[85] 李贤,钟仲贤.植物离体组织细胞染色体加倍技术在育种中的应用[J].上海农业学报,1998,14(3):99-102.
[86] 吴清,向素琼,严勇等.金荞麦的离体繁殖及同源四倍体的诱导[J].西南农业大学学报,2002,3(2):108-110.
[87] 常月梅.果树多倍体鉴定进展[J].山西林业科技,2000,3(l):l.
[88] 王丽艳,梁国鲁.植物多倍体的形成途径及鉴定方法[J].北方园艺,2004,(l):61.
[89] 郭启高,宋明,梁国鲁.植物多倍体诱导育种研究进展[J].生物学通报,2000,35(2):8.
[90] Sari N,Abak K,Pitrat M.Comparison of ploidylevel screening methods in watermelon: Citrullus lanatus (Thunb.) [J].Scientia Horticulturae,1999,82:265-277.
[91] Tambong J T,Sapra V T,Garton S.In vitro induction of tetraploids in colchicines-treated cocoyam plantlets [J].Euphytica,1998,104:191-197.
[92] Compton M,Barnett N,Gray D.Use of fluoresceindiacetate (FDA) to determine ploidy of invitro watermelon shoots [J].Plant Cell,Tissue and Organ Culture,1999,58:199-203.
[93] 刘文革,阉志红,饶小利.不同倍性西瓜的叶表皮微形态特征比较[J].果树学报,2005,22:31-34.
[94] Cohen D,Yao J.In vitro chromosome doubling of nine Zantedeschia cultivars [J].Plant Cell, Tissue and Organ Culture,1996,47:43-49.
[95] 杨瑞芳,郑思乡,郭清泉等.莲多倍体研究、莲花粉母细胞减数分裂行为观察[J].湖南农业大学学报,1998,24(2):95.
[96] 李愈学,张赞平.作物染色体及研究技术汇[M].北京:中国农业出版社,1990,10.
[97] 李步勋,陶抵辉,阮万辉.西瓜离体组织细胞染色体加倍技术的研究和应用[J].陕西农业科学,1999,(3):21.
[98] 秦盘山,王莉,陈素萍等.枸杞同源四倍侔的诱导与应用研究[A].自寿宁.宁夏枸杞研究[C].银川:宁夏人民出版社,1988:85-88.
[99] 艾先元,石巍峨,荆雅琴.枸杞茎尖培育四倍体苗初报[J].宁夏农林科技,I991.5-32.
[100] 王莉,陈紊萍,秦金山等.枸杞胚乳植株诱导和它的倍性水平[J].檀精学报,1985,12(6):440-444.
[101] 王莉,陈紊萍,秦金山等.枸杞胚乳植株的诱导及染色体倍性观察[J].中国科学(B辑),1986,690-606.
[102] 厦飒荣,桂耀林,棣廷玉.枸杞胚乳植株的诱导[J].植物学报,1885.27(1):106-109.
[103] 硕淑荣,桂耀韩.撩廷玉.枸杞胚乳植株的诱导及染色体倍性观察[A].自寿宁.宁夏枸杞研究[C].银川:宁夏人民出版社,1998:89-92.
[104] 马爱如,牛一恕.诱导宁夏拘杞多倍体研究初报[J].北农业科学,1987,6:26-27.
[105] 马爱如,牛一恕.三倍体宁夏枸杞研究初报[J].湖北农业科学,1988,9:31-32.
[106] 钟牲元,王燕,王锦秀.无耔拘杞选育初报[J].宁夏农林科技,1993,3:15-17.
[107] 安巍,李立精,焦忘宁等.三倍体无耔枸记新品种的选育研究[J].宁夏农林科技,1989,2:21-24.
[108] 张新宁,沈效东,王锦绣.枸杞四倍体同二倍体杂交败育的形态分析及解决方法[J].宁夏农林科技,1992(1):30-32.
[109] 厦飒荣,桂耀林,棣廷玉.枸杞胚乳植株的诱导[J].植物学报,1885,27(1):106-109.
[110] 陈紊萍,王莉,束秀清等.枸杞胚乳植株二代的形态及细胞学观察[A].自寿宁.宁夏枸杞研究[C].银川:宁夏人品出版社,1998,104-106.
[111] 王慧中,黄发蚰,李安生等.枸杞转基因植株的再生[A]白寿宁.宁夏枸杞研究[C]银川:宁夏人民出版社,1998:114-146.
[112] 王慧中,杜立群,黄发灿等.根瘤农杆菌介导的枸杞转化及转化植株的获得[J].中国科学(B辑),1993,23(4):391-395.
[113] Wu J H,Auline Moone.Totetraploid tangor plant regeneration from in vitro citrus somatic embryogensis callus treated with colchicines[J].Plant Cell Tissue and Organ Culture,2002,70:99-104.
[114] Yang X M,Cao Z Y,An L Z et al.In vitro tetraploid induction via colchicine treatment from diploid somatic embryos in grapevine ( Vitis vinifera L.) [J].Euphytica,2006,152:217–224.
[115] Murashige T,Skoog F.A revised medium for rapid growth and bioassays with tobacco tissue cultures [J].Plant Physiol,1962,15,473-497.
[116] Singer S R.Analysis growth in cell culture:calculating growth rates [J].Can J Bot,1986,64: 33-237.
[117] Fuentes S R L,Calheiros M B P,Manetti-Filho J et al.The effects of silver nitrate and different carbohydrate sources on somatic embryogenesis in Coffea canephora [J].Plant Cell Tiss Org Cult, 2000 (60):5-13.
[118] 崔凯荣,王晓哲,王亚馥.植物体细胞胚发生研究的一些现状[J].植物学通报,1993,10(3):14-20.
[119] Kong L,Yeung E C.Effects of ethylene and ethylene inhibitors on white spruce somatic embryo maturation [J].Plant Sci,1994(104):71-80.
[120] 杨汉民,高清祥,张爱国等.枸杞体细胞胚的诱导与形态发生[J].兰州大学学报,1992,28(1):87-95.
[121] Kadota M,Niimi Y.In vitro induction of tetraploid plants from a diploid Japanese pear cultivar (Pyrus pyrifolia N.cv.Hosui) [J].Plant Cell Rep,2002,21:282-286.
[122] Thao N T P,Ureshino K,Miyajima I et al.Induction of tetraploid in ornamental Alocasia through colchicine and oryzalin treatments [J].Plant Cell Tissue Organ Culture,2003,72:19-25.
[123] Michael M Blanke,Monika H?fer,Richard J.Pring Stomata and Structure of Tetraploid AppleLeaves cultured in Vitro Annals of Botany [J].Scientia Horticulturae,1994,73 (6):651-654.
[124] Chen L L,Gao S L.In vitro tetraploid induction and generation of tetraploids from mixoploids in Astragalus membranaceus [J].Plant Cell Rep,2001,112(3):339-344.
[125] 匡全,梁国鲁,郭启高等.秋水仙素诱导牛蒡多倍体[J].植物生理学通讯,2004,40(2): 157.
[126] Ollitroult Sammarcell.Ploidy levels in Kiwifrtui Plants determ inated by flow cytometer [J].Hort.Sci,1994,57(4):303-314.
[127] Costich D E Flow eytometer fou rapid determination of ploidy level in Vaceiniun [J].Theoretieal and Applied Geneties,1995,30(6):1276-1278.
[128] Leblanc O,Duenas M,Hemandez M et al.Chromosome doubling in Tripsacum:the production of artificial,sexual tetraploid plants [J].Plant-breeding,1995,114(3):226-230.