| 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-1containing0.03%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 for1020d, 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 Novembernext year January and AprilJuly. 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+H3BO350mg·L-1+CaCl220mg·L-1+agar3g·L-1for 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+H3BO3100mg·L-1+CaCl220mg·L-1+agar3g·L-1. The pollen viability was higher throughHAC and I2-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 at12days 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. |
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