【作者】 姜中武；

【导师】 束怀瑞； 陈学森；

【作者基本信息】 山东农业大学 ， 果树学， 2008， 博士

【摘要】 针对我国苹果主栽品种单一,自主知识产权优良品种贫乏,遗传基础狭窄,育种速度进展缓慢等现实问题,利用2n配子创造新的多倍体种质资源,对多倍体品种的生物学特性开展了广泛研究,其目的是为建立苹果高效多倍体育种体系,加快育种速度开展基础性研究工作,为多倍体品种的开发利用提供技术支撑。本研究的主要结果和结论如下:1、以“红将军”苹果作试材,对离体叶片诱导再生不定芽。结果表明:试管苗继代培养3~4代,当代培养25~30d,取顶部嫩叶3~4片,叶片近轴面向下接触诱导分化培养基,黑暗培养10~15d后,转入光照培养条件下,再生频率达100%。诱导红将军叶片高频再生的培养基组分是MS+TDZ 1.0mg/L+IAA 0.75 mg/L+蔗糖30g/L,培养基pH值5.8。采用浸渍法诱变叶片再生的秋水仙素的浓度以0.5%,处理时间48h,叶片诱变率为0.6%。混培法以40mg/L和50mg/L的秋水仙素浓度处理效果最好,诱变率分别为0.26%和0.37%。2、平邑甜茶具有很高的无融合生殖能力,但有少量的有性杂交后代,本试验检测了3054株,有性杂交率为4.7%。有性后代的四倍体植株的无融合特性的遗传,是平邑甜茶的雌2n配子的染色体组整体遗传的结果。石蜡切片发现,平邑甜茶的胚囊具有大孢子母细胞,并有二核胚囊、四核胚囊、八核胚囊各个发育期,但胚囊的发育成熟滞后于盛花期,平邑甜茶的雄花败育不形成小孢子(花粉)。平邑甜茶有部分大孢子母细胞殖可以减数分裂,减数后的胚囊与外源花粉杂交后,形成的三倍体。由于雌2n配子的形成滞后于花期,没有授粉机会的雌2n配子形成了无融合的三倍体种子,自然开放授粉的F1后代形成约2.5%的四倍体。可通过人工选择性杂交,在F1后代中选择四倍体的植株,测定无融合能力、抗逆性和嫁接亲合性,进行无融合砧木资源的创新。3、利用不同倍性的苹果品种进行杂交授粉,检测杂交F1代倍性分离规律,结果表明,不同倍性品种间杂交,F1代植株多为二倍体,比例在50%以上。四倍体天星×四倍体嘎啦杂交后代中二倍体、三倍体和四倍体的比例分别为18%、54%和18%。二倍体烟富4和四倍体花红杂交F1代中三倍体植株所占比例为47.6%。二倍体与二倍体杂交,三倍体比例约占2.5%;自然开放授粉三倍体后代低于0.25%。三倍体与三倍体品种杂交时可以授粉坐果,但是种子败育率较高。三倍体乔纳金与二倍体品种杂交,平均结籽率为2.5粒,杂交F1代中三倍体所占比例为28.4%。天星×四倍体嘎啦、烟富4×花红是培育三倍体品种的良好杂交组合。多倍体植株倍性不稳定,有自然二倍化的趋势,二倍体是苹果属最稳定的优化倍性状态。生产中应用的乔纳金、陆奥、北斗和北海道9号等三倍体品种都是由母本产生的雌2n配子形成的,染色体组整体源自母本。四倍体天星×四倍体嘎啦杂交三倍体后代染色体组的3个S基因型中的2个S基因是源自母本天星。4、不同倍性的花粉外观形态、叶绿素含量存在较大差别。不同倍性的苹果品种叶片叶绿素含量差异较大,叶绿素含量随着倍性的提高而降低。二倍体品种主要是三角型花粉,三倍体品种主要是圆型花粉,方型花粉可以作为多倍体的一个分级辅助指标。苹果栽培品种的三、四倍体都有花粉,可以做为授粉品种。花粉粒的含量与倍性无显著相关性。但无融合生殖类型的平邑甜茶、泰山海棠花药中无花粉粒存在。三倍体和四倍体品种花粉萌发孔区别较大,可将四孔花粉作为多倍性分类的辅助指标。三倍体的北海道9号和陆奥的应分划为N3-4P4C5类型。5、以乔纳金、王林、红富士作干砧,改接优良早熟苹果品种红露,结果表明:不同倍性干砧对红露苹果的果实硬度、含糖量和香气组成有较大影响。富士改接的红露苹果果实硬度最大,可溶性糖含量最高,而乔纳金上果实单果重最大,但硬度与可溶性糖含量最小;不同干砧能显著影响红露果实不同香气类别的含量,其中富士作干砧红露苹果中酯类和醇类含量最高,分别为4.56%和42.93%,乔纳金作干砧的红露果实酯类和醇类含量最低,分别为1.98%和29.76%。选用富士树高接,有利于提高红露果实硬度和风味品质。更多还原

【Abstract】 The aim of this study is to establish efficient apple polyploid breeding system and speed up apple breeding career that would work out for the existing problems of apple production in our country. The existing problems are mainly as follows: variety of main apple cultivar fewness, independent intellectual property rights species shot, genetic factor straitness and the progress of breeding tardiness , and so on. In order to exploit and utilize triploid cultivars, its biological characteristics had been investigated. The general idea included four steps. The first step is to select the best diploid cultivars as the excellence resource. The second step is to double it and make become tetraploid. Then the tetraploid across with different ploidy apple cultivars and obtain F1 hybrids. The last step is to determine the ploidy of hybrids by flow cytometry and AS-PCR. According to S-gen type , selecting young seedling and apple triploid cultivar breeding system.1、Experiments were carried out with new leaves of tissue culture plant of middle maturity cultivar Red General Apple (Malus domestica) to study the rate of adventitious bud regeneration. Regeneration rate have been studied by various plant growth regulator categories, concentration, darkness treatment time, and leaves part. The adventitious shoot were reproduced 3~4 generations in vitro , 3~4 top tender leaves of adventitious shoots were used as explant when the last subculture generation 25~35 days later . To cut the leaf edge off and press obverse leaf surface contact inducing medium. After 15 day darkness treatment, all treatment culture medium were moved to have light culture room, The adventitious bud regeneration rate were 100%. The optimal medium for regeneration is MS+TDZ1.0mg/L+IAA0.75 mg/L+Sucrose 30g/L , pH 5.8. The tetraploid plant were induced after the leaves were treated with the colchicine dipping for 48h, the percentages of tetraploid were 0.6%,with colchicine 40mg/L and 50mg/L on mix medium treatment the percentages of tretaploid is 0.26% and 0.37%,respectively.2、Although M.hupehensis ( Pamp. ) Rehd.var.pinyyiensis.Jiang has apomixi procreate ability, it still has hybrids. Our results showed that the rate of hybrid was 4.7 percent. The apospory may meiosis to become bivalence or trivalence, then by hybridize with foreign pollen to produce triploid or tetraploid hybrids. And these hybrids could keep the characteristic of apomixi of which was the result of whole hereditary of female unreduced gamete. But the diploid hybrids has not apomixi procreate ability. The result of paraffin slice up showed that the ovule of M. var.pinyyiensis.Jiang has MMC, we also found two-nucleus megaspore, four-nucleus megaspore and eight-nucleus megaspore. But the growth of megaspore lagged the florescence. And the flower has not pollen. Because the developing period of unreduced gamete lagged bloom, some unreduced gamete that hadn’t been pollination developped apomixi procreated triploid seed, about 2.5 percent F1 hybrids which had been pollinated developped tetraploid offspring. therefore,we may hybridize different apple resource purposively and get the innovation of rootstock resource by selectting and mensurating the apomixi procreate ability of tetraploid offspring.3、To investigate the cross-compatibility in different ploidy apple cultivars, the ploidy of F1 hybrids had been detected by flow cytometry . The result showed that over 50 percent F1 offspring is diploid. And many of polyploid plant is unstable which would become diploid in growing process. The state of diploid is the most stable ploidy in apple species. We studied the ploidy of hybrids of Tetraploid Tianxing and Tetraploid Royal Gala , the result showed that the ratio of diploid and tetraploid is 18 percent, the rest being triploid. The hybrid of diploid and diploid can form a little triploid , the percentation was about 2.5 percent, but triploid percentation is lower than 0.25%. When triploid crossed with triploid , the fruit could been formed, but the seed often cease growth easily. Nevertheless, when crossed with diploid, the seed set was higher, and the average of seed is 2.5 per fruit.Many of triploid apple cultivars, such as Jonagold, Hokato, Mustu and Hac9 etc, derived from female unreduced gamete. Two S-gene of triploid hybrids obtained from Tianxing and Royal Gala come from Tianxing by AS-PCR. Tianxing and Royal Gala are more compatible to breed triploid cultivars. 4、The pollen shape and the chlorophyll content of different ploidy apple cultivars have obvious difference. With the decrease of ploidy, the chlorophyll content is increase, nevertheless the growth amount is decrease. The amount of pollen of every anther isn’t orderliness. And there isn’t different between the triploid and diploid cultivars. According to the number of pollen aperture, tetraploid and tripoid have obvious difference with diploid. Such as Hokato and Hac9 are N3-4P4C5 type.5、Method of body interstocks, head space-solid phase microextraction (HS-SPME) and gas chromatography-mass spectrometry (GC-MS) were used to measure the composing factors of fruit quality and aroma compound of Honglu fruit on different cultivars body interstocks. The results showed that the firmness (7.02kg/cm2) and soluble solid sugar content (12.5%) of Honglu fruit on Fuji body graft were the highest compared with the fruit on other body graft. However, the firmness and SSC of Honglu fruit on Jonagold body interstock were the lowest, but its fruit weight was the highest (211.85g). The body interstock could not affect the number of aroma compounds of Honglu, but it could affect kinds of aroma significantly. The relative content of the same aroma category of Honglu fruit was different on different cultivars body interstocks. The contents of esters and alcohols were the highest (4.56% and 42.93%, respectively) on Fuji interstock, and they were the lowest on Jonagold body graft, 1.98% and 29.76% respectively. So grafting on Fuji body interstock could enhance the fruit quality of Honglu apple.更多还原