君子兰多倍体诱导及种间杂交亲和性研究
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摘要
君子兰原产南非，为石蒜科（Amaryllidaceae）君子兰属（Clivia）多年生常绿草本植物，是重要室内盆栽花卉。君子兰属约有7个种，常见栽培种为大花君子兰（C.miniata）。本文采用秋水仙素结合组织培养法对大花君子兰未成熟胚和种子进行离体四倍体诱导，得到了加倍植株，并对其染色体数目、叶片及气孔特性、叶绿素含量进行观察。同时，以大花君子兰（C. miniata）、黄花君子兰（C. miniata var. citrina）和垂笑君子兰（C. nobilis）为试材，观察其花粉母细胞减数分裂、测定花粉生活力及柱头可授性，并进行种及变种间正反交及自交，利用荧光显微镜观察花粉萌发及花粉管伸长情况，研究了君子兰种及变种间杂交亲和性，得到了种间杂种实生苗。主要结果如下：
采用秋水仙素离体诱导大花君子兰品种‘胜利’的未成熟胚，研究不同秋水仙素浓度、处理时间、胚龄、预培养、低温处理对未成熟胚的加倍诱导。结果表明，以未成熟胚为外植体进行秋水仙素多倍体诱导，平均四倍体诱导率为11.7%。秋水仙素浓度、处理时间、胚龄共同影响诱导效果，低浓度短时间、高浓度长时间均不适合君子兰多倍体诱导。随秋水仙素浓度增加，诱导率增加，但浓度超过0.03%，死亡率大大增加，四倍体诱导率降低。在MS+NAA2.0mg·L~(-1)+BA1.5mg·L~(-1)培养基上，120d、150d和180d胚龄的未成熟胚均可诱导加倍，但诱导率不同，胚龄为120d时，0.03%秋水仙素处理30d，胚的死亡率为46.7%，四倍体诱导率为20.9%；胚龄为180d时，0.03%秋水仙素处理20d，四倍体诱导率为30.0%，诱导效果最好。预培养增加未成熟胚对秋水仙素的抗性，不利于多倍体诱导，预培养20d，0.01%秋水仙素处理后胚的死亡率和形态变化率均为0。低温预处理的未成熟胚对秋水仙素更敏感，形态变化率增加。君子兰最佳生根培养基为1/2MS+NAA1.5mg·L~(-1)+活性炭2.0g·L~(-1)+蔗糖10g·L~(-1)+琼脂5g·L~(-1)，60d后生根率达72.2%，单株根数为2.9条，而经秋水仙素加倍处理的株系生根周期长、生根率低、单株生根数少。
采用秋水仙素离体诱导大花君子兰品种‘胜利’和‘缟兰’种子，研究不同秋水仙素浓度、处理时间、预培养和品种对种子的加倍存活及形态变化的影响。结果表明，秋水仙素处理的种子，多数可正常萌发和生长，无形态变化；一些种子萌发出的胚芽鞘肥大、变厚、生长慢，但继续培养后慢慢消失；一些种子会褐化或死亡，0.03%秋水仙素处理30d时，死亡率为25.5%。预培养10~20d后种子萌发，会增加种胚对秋水仙素的吸收，但0.01%秋水仙素处理下，未获得加倍植株。
对秋水仙素诱导的加倍植株进行细胞学、形态学及生理指标鉴定。结果表明，秋水仙素诱导未成熟胚加倍处理后代中同时存在2x、4x和混倍体植株，共获得37株四倍体（4n=4x=44）、210株二倍体（2n=2x=22）和68株混倍体植株。秋水仙素诱导的四倍体君子兰生长缓慢、生根明显较晚、叶色浓绿、叶片明显变厚（1.35mm）、变宽（3.20cm）、变短（7.85cm）、叶形指数变小（2.52）。四倍体植株的气孔和保卫细胞变大，其中气孔长度增加42.8%、保卫细胞长度和宽度分别增加49.3%和36.4%，气孔密度降低50.6%，保卫细胞内叶绿体数目增加到41.2个。不同倍性君子兰的叶绿素含量无显著差异。因此，叶片和气孔性状可以作为早期判断君子兰是否加倍的辅助指标。
调查3种君子兰的开花物候期，观察花粉母细胞减数分裂进程与花蕾长度的关系及减数分裂行为。结果表明，3种君子兰中，大花君子兰和黄花君子兰花期主要集中在春季（1月~3月）；垂笑君子兰有2次花期，主要集中在冬季和春夏季（11月~翌年1月末和4~7月）。大花君子兰和黄花君子兰的花蕾长度在0.4~1.0cm为花粉母细胞减数分裂时期，长度在0.8~1.0cm时，已形成四分体或花粉粒；垂笑君子兰花蕾长度在0.35~0.6cm为花粉母细胞减数分裂时期，其持续时间较短、分裂快，长度大于0.6cm已发育成花粉粒。
利用扫描电镜和光学显微镜观察3种君子兰的花粉形态、花粉生活力及柱头可授性。结果表明，3种君子兰花粉形态相似，极面观为椭圆形，赤道面观近肾形，其中，黄花君子兰花粉最大、最圆；垂笑君子兰花粉最小、最扁。大花君子兰和黄花君子兰花粉生活力较高，分别为81.8%和82.6%，花粉离体萌发最佳培养基为蔗糖100g·L~(-1)+H_3BO_350mg·L~(-1)+CaCl_220mg·L~(-1)+琼脂3g·L~(-1)；而垂笑君子兰花粉生活力仅为42.6%，最佳培养基为蔗糖100g·L~(-1)+H_3BO_3100mg·L~(-1)+CaCl_230mg·L~(-1)+琼脂3g·L~(-1)。HAC和I_2-KI染色法所测花粉生活力均略高于培养基法；而TTC染色法未能使花粉染色，不适宜测定君子兰花粉。常温贮藏花粉生活力下降较快，大花君子兰的花粉贮藏30d后的生活力由81.8%降到29.6%，垂笑君子兰花粉贮藏20d后的生活力由42.6%降到10.9%；4℃低温和冷冻贮藏花粉生活力下降较慢，低温贮藏120d后大花君子兰和垂笑君子兰的花粉生活力分别为32.1%和15.2%；冷冻贮藏120d后分别为52.7%和20.3%。3种君子兰在开花当天采集的花粉萌发率最高，开花后1～2d内柱头可授性最高。
对君子兰3个种及变种进行种间正反交，并利用荧光显微镜观察花粉萌发及花粉管伸长情况，研究不同杂交组合的亲和性。结果表明，君子兰3个种及变种间正反交均可得到少量杂交果实并获得有胚种子，但坐果率和单果种子数都低于自交。其中，垂笑君子兰与大花君子兰和黄花君子兰杂交坐果率最低，分别为15.9%和19.1%，单果种子数最少，分别为1.2个和1.4个，杂交亲和性最差，但反交组合坐果率则稍高，亲和性高于以垂笑君子兰为母本进行种间杂交。大花君子兰与其变种黄花君子兰的杂交亲和性强、亲缘关系较近，正反交的坐果率和单果种子数均较高，分别为68.8%和57.5%，5.3个和6.5个。大花君子兰和黄花君子兰正反交成熟果实最大；以垂笑君子兰为父本与其种间杂交的果实稍小；而以垂笑君子兰为母本进行种间杂交的果实最小，圆球形。采用生长调节物质涂抹柱头和重复授粉，均可大大提高种间杂交的坐果率，但单果种子数变化很小，而切割柱头和花柱未能提高君子兰杂交坐果率。
荧光观察发现，君子兰种间杂交的花粉萌发与花粉管伸长均比自交明显滞后，花粉管和柱头中均出现大量胼胝质，阻碍了花粉萌发和花粉管的伸长，而自交组合中乳突细胞表面未观察到胼胝质。以垂笑君子兰为父本种间杂交时，其花粉在柱头上萌发较晚、花粉管伸长较慢、花粉管中均出现大量胼胝质，只有少量花粉管可以穿过花柱、进入子房。此外，还观察到花粉管缠绕在柱头上呈螺旋状伸长、花粉管在伸长过程中出现回转等异常现象。以垂笑君子兰为母本种间杂交中，柱头乳突细胞表面均出现较多胼胝质，阻碍花粉萌发和花粉管向下伸长。
Clivia miniata Regel., a perennial herbaceous flower belonging to the genus Clivia inthe Amaryllidaceae, is native to South African. Clivia is an important indoor potted flower.Up to now, the genus Clivia comprises seven recognized species, in which C. miniata(2n=2x=22) is the most commonly cultivated species in the world. In this paper, the C.miniata were treated via colchicine combined with culture in vitro to induce tetraploids andthe chromosome count, leaf and stoma characteristics, chlorophyll content were identifiedand compared with diploids, some tetraploids were obtained. Simultaneously, the threespecies or varieties, including C. miniata, C. miniata var. citrina and C. nobilis, were used asexperimental materials to carry out interspecific reciprocal crosses and selfing. The pollenmother cells meiosis, pollen shape and viability and stigma receptivity were observed byoptical microscope and scan microscope, and the pollen and pollen tube growth wereobserved by fluorescence microscope to study the compatibility of interspecific hybridization,some seedlings of interspecific hybrid were obtained. The main results were as follows.
The immature embryos of C. miniata ‘Shengli’were induced through colchicine in vitro,the effects of different colchicine concentrations, durations, embryo ages, pre-culturedurations, low temperature treatment durations on doubling induction of embryos werestudied. The results showed that there was an average induction rate of11.7%when immatureembryos were used as explants to induce tetraploids in vitro. Colchicine concentration,duration and embryo age were key factors, lower colchicine concentration combined withshorter duration or higher colchicine concentration combined with longer duration were notsuitable for polyploidy induction in Clivia. The induction rate of tetraploid increased with theincrease of colchicine concentration, but decreased when the colchicine concentration wasabove0.03%, with higher death rate. The tetraploids could be obtained from immatureembryos of120d,150d and180d embryo age, but culturing120d immature embryos for30d on MS+NAA2.0mg·L~(-1)+BA1.5mg·L~(-1)containing0.03%colchicine with death rate of46.7%and induction rate of20.9%; culturing180d immature embryos for20d with0.03%colchicine was the most effective treatment with the highest induction rate of30.0%. Afterpre-culturing for some days on the medium without colchicine, the immature embryos wereless sensitive to colchicine, the death rate and morphological change rate of embryos were0when pre-cultured for20d on the medium containing0.01%colchicine. But the immatureembryos were more sensitive to colchicine after low temparature treatment, themorphological change rate were increased. The most effective rooting medium was1/2MS+NAA1.5mg·L~(-1)+AC2.0g·L~(-1)+sucrose10g·L~(-1)+agar5g·L~(-1), the rooting rate and number of roots per plantlet were72.2%and2.9respectively after culturing for60d. But the plantletscolchicine-induced had longer rooting cycle, lower rooting rate and roots per plantlet.
The seeds of C. miniata cvs ‘Shengli’ and ‘Gaolan’ were induced via colchicine in vitro,the effects of different colchicine concentrations, durations, pre-cultures and varieties on thesurvival and morphological change of seeds were studied. The results showed that the seedswere not sensitive to colchicine, a lot of seeds colchicine-treated could germinate and grownormally without morphological change; a few coleoptiles of germinated seeds becamehypertrophy, thicker and slower growth, but disappeared after culturing for some days on themedium without colchicine; a few seeds became brown or died, the death rate was25.5%when treated with0.03%colchicine for30d. Germinated seeds were more sensitive tocolchicine after pre-culturing for10~20d, but no tetraploids were obtained through0.01%colchicine, maybe lower colchicine concentration.
The cytological, morphological characteristics and physiological indexes ofcolchicine-induced plantlets were identified and observed. The results showed that37tetraploids (4n=4x=44),210diploids (2n=2x=22) and68mixoploids were obtained fromcolchicine-induced plantlets. The tetraploids colchicine-induced had a slower growth, laterrooting, darker leaf color, and the leaf became thicker (1.35mm), wider (3.20cm) and shorter(7.85cm), the leaf index became smaller (2.52). Compared with diploids, the tetraploids hadlarger size of stomata and guard cells, the length of stomata increased42.8%, the stomatadensity decreased50.6%, and the length and width of guard cells increased49.3%and36.4%respectively, the chloroplasts per pair of guard cells increased to41.21, but there was nodifference in chlorophyll content of tetraploids and diploids. So, the leaf and stomacharacteristics could be used for auxiliary screening indexes of tetraploids in early period ofC. miniata.
The blooming phenophases of three Clivia species were investigated, the correlations ofmeiosis progress and bud lengh, and the meiosis progress of pollen mother cells wereobserved. The results showed that among three Clivia species, the florescence of C. miniataand C. miniata var. citrina was mainly from January to March, but C. nobilis had twoflorescences every year, mainly from November~next year January and April~July. Pollenmother cells of C. miniata and C. miniata var. citrina were in meiosis period when their budlength was between0.4cm and1.0cm, tetrahedral tetrad or pollen grain were formed whenthe bud length was between0.8cm and1.0cm. Pollen mother cells of C. nobilis were inmeiosis period when the bud length was between0.35cm and0.6cm, which was short, andpollen grains were formed when the bud length was above0.6cm.
The pollen shape, pollen viability and stigma receptivity of three Clivia species were observed via scanning electron microscope and optical microscope. The results indicated thatthe pollen shape of three Clivia were similar, pollen was oval in polar view, and reniform inequatorial view. Of them, the pollen of C. miniata var. citrina was the biggest and rounddest,the C. nobilis was the smallest and flattest. The effective germination medium of pollen wassugar100g·L~(-1)+H_3BO_350mg·L~(-1)+CaCl_220mg·L~(-1)+agar3g·L~(-1)for C. miniata and C.miniata var. citrina, with the pollen viability of81.8%and82.6%respectively. The pollenviability of C. nobilis was the lowest of42.6%, the effective medium was sugar100g·L~(-1)+H_3BO_3100mg·L~(-1)+CaCl_220mg·L~(-1)+agar3g·L~(-1). The pollen viability was higher throughHAC and I_2-KI straining method than cultured in vitro, but TTC was not suitable for Clivia.The germinate rate of pollen storaged at room temperature decreased faster than at lowtemperature and freeze temperature. The pollen viability of C. miniata and C. nobilisdecreased from81.8%to29.6%and from42.6%to10.9%after storaged for30d at roomtemperature; the pollen viability of C. miniata and C. nobilis decreased to32.1%and15.2%respectively after120d at4℃low temperature; the pollen viability decreased to52.7%and20.3%respectively after120d at freeze temperature. Of three Clivia species, the germinationrate of pollen collected at the bloom day was the highest, the stigma receptivity was thehighest at1~2days after bloom.
The three Clivia species or varieties were used as materials to carry out interspecificreciprocal crosses and selfing, and the pollen germination and pollen tube behavior afterpollination were observed to study the compatibility via fluorescence microscope. The resultsindicated that a few fruits and seeds with embryo were obtained through interspecifichybridization, but the fruit setting rate and number of seeds per fruit were less than that ofselfing. Of them, the fruit setting rate and number of seeds per fruit were the lowest of15.9%and19.1%,1.2and1.4respectively in the cross of C. nobilis×C. miniata and C. nobilis×C.miniata var. citrina, but the fruit setting rate were higher in their inverse cross combinations.So, when the C. nobilis was used as male parent, the cross-compatibility was higher than usedas female parent. The fruit setting rate of reciprocal crosses between C. miniata and C.miniata var. citrina was68.8%and57.5%respectively, and the seeds per fruit reached5.3and6.5respectively, which showed the compatibility was the best, genetic relationship wasthe closest. The mature fruits of reciprocal crosses between C. miniata and C. miniata var.citrina were the bigest, the fruits when C. nobilis was used as male parent in interspecifichybridization were smaller, but the smallest and spheroidal in their inverse crosscombinations. Repeated-pollination and regulators disposal stigma could greatly improve thefruit setting rate in interspecific crosses of Clivia, but the number of seeds per fruit had fewchanges. Cutting-style pollination could not improve fruit setting rate.
In contrast to selfing of Clivia, the pollens germinated later and pollen tube elongatedslower obviously in interspecific crosses of Clivia via fluorescence microscope, and callosesin the pollen tubes and on the stigmas hindered pollen germination and pollen tube elongation.The pollens of C. nobilis in interspecific crosses germinated later and elongated slowerobviously, many calloses occurred in pollen tube and few pollen tubes reached ovary within96h after pollination, in addition, some abnormal phenomenons were observed, such asspiraling pollen tube around papilla cells on stigma, gyration of pollen tube and so on. Thecalloses on the stigmas hindered pollen germination when C. nobilis was used as the femaleparent.
采用秋水仙素离体诱导大花君子兰品种‘胜利’的未成熟胚，研究不同秋水仙素浓度、处理时间、胚龄、预培养、低温处理对未成熟胚的加倍诱导。结果表明，以未成熟胚为外植体进行秋水仙素多倍体诱导，平均四倍体诱导率为11.7%。秋水仙素浓度、处理时间、胚龄共同影响诱导效果，低浓度短时间、高浓度长时间均不适合君子兰多倍体诱导。随秋水仙素浓度增加，诱导率增加，但浓度超过0.03%，死亡率大大增加，四倍体诱导率降低。在MS+NAA2.0mg·L~(-1)+BA1.5mg·L~(-1)培养基上，120d、150d和180d胚龄的未成熟胚均可诱导加倍，但诱导率不同，胚龄为120d时，0.03%秋水仙素处理30d，胚的死亡率为46.7%，四倍体诱导率为20.9%；胚龄为180d时，0.03%秋水仙素处理20d，四倍体诱导率为30.0%，诱导效果最好。预培养增加未成熟胚对秋水仙素的抗性，不利于多倍体诱导，预培养20d，0.01%秋水仙素处理后胚的死亡率和形态变化率均为0。低温预处理的未成熟胚对秋水仙素更敏感，形态变化率增加。君子兰最佳生根培养基为1/2MS+NAA1.5mg·L~(-1)+活性炭2.0g·L~(-1)+蔗糖10g·L~(-1)+琼脂5g·L~(-1)，60d后生根率达72.2%，单株根数为2.9条，而经秋水仙素加倍处理的株系生根周期长、生根率低、单株生根数少。
采用秋水仙素离体诱导大花君子兰品种‘胜利’和‘缟兰’种子，研究不同秋水仙素浓度、处理时间、预培养和品种对种子的加倍存活及形态变化的影响。结果表明，秋水仙素处理的种子，多数可正常萌发和生长，无形态变化；一些种子萌发出的胚芽鞘肥大、变厚、生长慢，但继续培养后慢慢消失；一些种子会褐化或死亡，0.03%秋水仙素处理30d时，死亡率为25.5%。预培养10~20d后种子萌发，会增加种胚对秋水仙素的吸收，但0.01%秋水仙素处理下，未获得加倍植株。
对秋水仙素诱导的加倍植株进行细胞学、形态学及生理指标鉴定。结果表明，秋水仙素诱导未成熟胚加倍处理后代中同时存在2x、4x和混倍体植株，共获得37株四倍体（4n=4x=44）、210株二倍体（2n=2x=22）和68株混倍体植株。秋水仙素诱导的四倍体君子兰生长缓慢、生根明显较晚、叶色浓绿、叶片明显变厚（1.35mm）、变宽（3.20cm）、变短（7.85cm）、叶形指数变小（2.52）。四倍体植株的气孔和保卫细胞变大，其中气孔长度增加42.8%、保卫细胞长度和宽度分别增加49.3%和36.4%，气孔密度降低50.6%，保卫细胞内叶绿体数目增加到41.2个。不同倍性君子兰的叶绿素含量无显著差异。因此，叶片和气孔性状可以作为早期判断君子兰是否加倍的辅助指标。
调查3种君子兰的开花物候期，观察花粉母细胞减数分裂进程与花蕾长度的关系及减数分裂行为。结果表明，3种君子兰中，大花君子兰和黄花君子兰花期主要集中在春季（1月~3月）；垂笑君子兰有2次花期，主要集中在冬季和春夏季（11月~翌年1月末和4~7月）。大花君子兰和黄花君子兰的花蕾长度在0.4~1.0cm为花粉母细胞减数分裂时期，长度在0.8~1.0cm时，已形成四分体或花粉粒；垂笑君子兰花蕾长度在0.35~0.6cm为花粉母细胞减数分裂时期，其持续时间较短、分裂快，长度大于0.6cm已发育成花粉粒。
利用扫描电镜和光学显微镜观察3种君子兰的花粉形态、花粉生活力及柱头可授性。结果表明，3种君子兰花粉形态相似，极面观为椭圆形，赤道面观近肾形，其中，黄花君子兰花粉最大、最圆；垂笑君子兰花粉最小、最扁。大花君子兰和黄花君子兰花粉生活力较高，分别为81.8%和82.6%，花粉离体萌发最佳培养基为蔗糖100g·L~(-1)+H_3BO_350mg·L~(-1)+CaCl_220mg·L~(-1)+琼脂3g·L~(-1)；而垂笑君子兰花粉生活力仅为42.6%，最佳培养基为蔗糖100g·L~(-1)+H_3BO_3100mg·L~(-1)+CaCl_230mg·L~(-1)+琼脂3g·L~(-1)。HAC和I_2-KI染色法所测花粉生活力均略高于培养基法；而TTC染色法未能使花粉染色，不适宜测定君子兰花粉。常温贮藏花粉生活力下降较快，大花君子兰的花粉贮藏30d后的生活力由81.8%降到29.6%，垂笑君子兰花粉贮藏20d后的生活力由42.6%降到10.9%；4℃低温和冷冻贮藏花粉生活力下降较慢，低温贮藏120d后大花君子兰和垂笑君子兰的花粉生活力分别为32.1%和15.2%；冷冻贮藏120d后分别为52.7%和20.3%。3种君子兰在开花当天采集的花粉萌发率最高，开花后1～2d内柱头可授性最高。
对君子兰3个种及变种进行种间正反交，并利用荧光显微镜观察花粉萌发及花粉管伸长情况，研究不同杂交组合的亲和性。结果表明，君子兰3个种及变种间正反交均可得到少量杂交果实并获得有胚种子，但坐果率和单果种子数都低于自交。其中，垂笑君子兰与大花君子兰和黄花君子兰杂交坐果率最低，分别为15.9%和19.1%，单果种子数最少，分别为1.2个和1.4个，杂交亲和性最差，但反交组合坐果率则稍高，亲和性高于以垂笑君子兰为母本进行种间杂交。大花君子兰与其变种黄花君子兰的杂交亲和性强、亲缘关系较近，正反交的坐果率和单果种子数均较高，分别为68.8%和57.5%，5.3个和6.5个。大花君子兰和黄花君子兰正反交成熟果实最大；以垂笑君子兰为父本与其种间杂交的果实稍小；而以垂笑君子兰为母本进行种间杂交的果实最小，圆球形。采用生长调节物质涂抹柱头和重复授粉，均可大大提高种间杂交的坐果率，但单果种子数变化很小，而切割柱头和花柱未能提高君子兰杂交坐果率。
荧光观察发现，君子兰种间杂交的花粉萌发与花粉管伸长均比自交明显滞后，花粉管和柱头中均出现大量胼胝质，阻碍了花粉萌发和花粉管的伸长，而自交组合中乳突细胞表面未观察到胼胝质。以垂笑君子兰为父本种间杂交时，其花粉在柱头上萌发较晚、花粉管伸长较慢、花粉管中均出现大量胼胝质，只有少量花粉管可以穿过花柱、进入子房。此外，还观察到花粉管缠绕在柱头上呈螺旋状伸长、花粉管在伸长过程中出现回转等异常现象。以垂笑君子兰为母本种间杂交中，柱头乳突细胞表面均出现较多胼胝质，阻碍花粉萌发和花粉管向下伸长。
Clivia miniata Regel., a perennial herbaceous flower belonging to the genus Clivia inthe Amaryllidaceae, is native to South African. Clivia is an important indoor potted flower.Up to now, the genus Clivia comprises seven recognized species, in which C. miniata(2n=2x=22) is the most commonly cultivated species in the world. In this paper, the C.miniata were treated via colchicine combined with culture in vitro to induce tetraploids andthe chromosome count, leaf and stoma characteristics, chlorophyll content were identifiedand compared with diploids, some tetraploids were obtained. Simultaneously, the threespecies or varieties, including C. miniata, C. miniata var. citrina and C. nobilis, were used asexperimental materials to carry out interspecific reciprocal crosses and selfing. The pollenmother cells meiosis, pollen shape and viability and stigma receptivity were observed byoptical microscope and scan microscope, and the pollen and pollen tube growth wereobserved by fluorescence microscope to study the compatibility of interspecific hybridization,some seedlings of interspecific hybrid were obtained. The main results were as follows.
The immature embryos of C. miniata ‘Shengli’were induced through colchicine in vitro,the effects of different colchicine concentrations, durations, embryo ages, pre-culturedurations, low temperature treatment durations on doubling induction of embryos werestudied. The results showed that there was an average induction rate of11.7%when immatureembryos were used as explants to induce tetraploids in vitro. Colchicine concentration,duration and embryo age were key factors, lower colchicine concentration combined withshorter duration or higher colchicine concentration combined with longer duration were notsuitable for polyploidy induction in Clivia. The induction rate of tetraploid increased with theincrease of colchicine concentration, but decreased when the colchicine concentration wasabove0.03%, with higher death rate. The tetraploids could be obtained from immatureembryos of120d,150d and180d embryo age, but culturing120d immature embryos for30d on MS+NAA2.0mg·L~(-1)+BA1.5mg·L~(-1)containing0.03%colchicine with death rate of46.7%and induction rate of20.9%; culturing180d immature embryos for20d with0.03%colchicine was the most effective treatment with the highest induction rate of30.0%. Afterpre-culturing for some days on the medium without colchicine, the immature embryos wereless sensitive to colchicine, the death rate and morphological change rate of embryos were0when pre-cultured for20d on the medium containing0.01%colchicine. But the immatureembryos were more sensitive to colchicine after low temparature treatment, themorphological change rate were increased. The most effective rooting medium was1/2MS+NAA1.5mg·L~(-1)+AC2.0g·L~(-1)+sucrose10g·L~(-1)+agar5g·L~(-1), the rooting rate and number of roots per plantlet were72.2%and2.9respectively after culturing for60d. But the plantletscolchicine-induced had longer rooting cycle, lower rooting rate and roots per plantlet.
The seeds of C. miniata cvs ‘Shengli’ and ‘Gaolan’ were induced via colchicine in vitro,the effects of different colchicine concentrations, durations, pre-cultures and varieties on thesurvival and morphological change of seeds were studied. The results showed that the seedswere not sensitive to colchicine, a lot of seeds colchicine-treated could germinate and grownormally without morphological change; a few coleoptiles of germinated seeds becamehypertrophy, thicker and slower growth, but disappeared after culturing for some days on themedium without colchicine; a few seeds became brown or died, the death rate was25.5%when treated with0.03%colchicine for30d. Germinated seeds were more sensitive tocolchicine after pre-culturing for10~20d, but no tetraploids were obtained through0.01%colchicine, maybe lower colchicine concentration.
The cytological, morphological characteristics and physiological indexes ofcolchicine-induced plantlets were identified and observed. The results showed that37tetraploids (4n=4x=44),210diploids (2n=2x=22) and68mixoploids were obtained fromcolchicine-induced plantlets. The tetraploids colchicine-induced had a slower growth, laterrooting, darker leaf color, and the leaf became thicker (1.35mm), wider (3.20cm) and shorter(7.85cm), the leaf index became smaller (2.52). Compared with diploids, the tetraploids hadlarger size of stomata and guard cells, the length of stomata increased42.8%, the stomatadensity decreased50.6%, and the length and width of guard cells increased49.3%and36.4%respectively, the chloroplasts per pair of guard cells increased to41.21, but there was nodifference in chlorophyll content of tetraploids and diploids. So, the leaf and stomacharacteristics could be used for auxiliary screening indexes of tetraploids in early period ofC. miniata.
The blooming phenophases of three Clivia species were investigated, the correlations ofmeiosis progress and bud lengh, and the meiosis progress of pollen mother cells wereobserved. The results showed that among three Clivia species, the florescence of C. miniataand C. miniata var. citrina was mainly from January to March, but C. nobilis had twoflorescences every year, mainly from November~next year January and April~July. Pollenmother cells of C. miniata and C. miniata var. citrina were in meiosis period when their budlength was between0.4cm and1.0cm, tetrahedral tetrad or pollen grain were formed whenthe bud length was between0.8cm and1.0cm. Pollen mother cells of C. nobilis were inmeiosis period when the bud length was between0.35cm and0.6cm, which was short, andpollen grains were formed when the bud length was above0.6cm.
The pollen shape, pollen viability and stigma receptivity of three Clivia species were observed via scanning electron microscope and optical microscope. The results indicated thatthe pollen shape of three Clivia were similar, pollen was oval in polar view, and reniform inequatorial view. Of them, the pollen of C. miniata var. citrina was the biggest and rounddest,the C. nobilis was the smallest and flattest. The effective germination medium of pollen wassugar100g·L~(-1)+H_3BO_350mg·L~(-1)+CaCl_220mg·L~(-1)+agar3g·L~(-1)for C. miniata and C.miniata var. citrina, with the pollen viability of81.8%and82.6%respectively. The pollenviability of C. nobilis was the lowest of42.6%, the effective medium was sugar100g·L~(-1)+H_3BO_3100mg·L~(-1)+CaCl_220mg·L~(-1)+agar3g·L~(-1). The pollen viability was higher throughHAC and I_2-KI straining method than cultured in vitro, but TTC was not suitable for Clivia.The germinate rate of pollen storaged at room temperature decreased faster than at lowtemperature and freeze temperature. The pollen viability of C. miniata and C. nobilisdecreased from81.8%to29.6%and from42.6%to10.9%after storaged for30d at roomtemperature; the pollen viability of C. miniata and C. nobilis decreased to32.1%and15.2%respectively after120d at4℃low temperature; the pollen viability decreased to52.7%and20.3%respectively after120d at freeze temperature. Of three Clivia species, the germinationrate of pollen collected at the bloom day was the highest, the stigma receptivity was thehighest at1~2days after bloom.
The three Clivia species or varieties were used as materials to carry out interspecificreciprocal crosses and selfing, and the pollen germination and pollen tube behavior afterpollination were observed to study the compatibility via fluorescence microscope. The resultsindicated that a few fruits and seeds with embryo were obtained through interspecifichybridization, but the fruit setting rate and number of seeds per fruit were less than that ofselfing. Of them, the fruit setting rate and number of seeds per fruit were the lowest of15.9%and19.1%,1.2and1.4respectively in the cross of C. nobilis×C. miniata and C. nobilis×C.miniata var. citrina, but the fruit setting rate were higher in their inverse cross combinations.So, when the C. nobilis was used as male parent, the cross-compatibility was higher than usedas female parent. The fruit setting rate of reciprocal crosses between C. miniata and C.miniata var. citrina was68.8%and57.5%respectively, and the seeds per fruit reached5.3and6.5respectively, which showed the compatibility was the best, genetic relationship wasthe closest. The mature fruits of reciprocal crosses between C. miniata and C. miniata var.citrina were the bigest, the fruits when C. nobilis was used as male parent in interspecifichybridization were smaller, but the smallest and spheroidal in their inverse crosscombinations. Repeated-pollination and regulators disposal stigma could greatly improve thefruit setting rate in interspecific crosses of Clivia, but the number of seeds per fruit had fewchanges. Cutting-style pollination could not improve fruit setting rate.
In contrast to selfing of Clivia, the pollens germinated later and pollen tube elongatedslower obviously in interspecific crosses of Clivia via fluorescence microscope, and callosesin the pollen tubes and on the stigmas hindered pollen germination and pollen tube elongation.The pollens of C. nobilis in interspecific crosses germinated later and elongated slowerobviously, many calloses occurred in pollen tube and few pollen tubes reached ovary within96h after pollination, in addition, some abnormal phenomenons were observed, such asspiraling pollen tube around papilla cells on stigma, gyration of pollen tube and so on. Thecalloses on the stigmas hindered pollen germination when C. nobilis was used as the femaleparent.
引文
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