【作者】 唐懿；

【导师】 李焕秀；

【作者基本信息】 四川农业大学 ， 果树学， 2011， 博士

【摘要】 苦瓜为葫芦科苦瓜属植物,它不仅是我国及东南亚各国人民喜爱的传统蔬菜,也是一种重要的药用植物。花药培养是获得单倍体植株最有效的方法之一,对植物育种有着非常重要的意义。本试验对影响苦瓜花药培养的诸因素进行了较系统的研究,首次从苦瓜花药愈伤组织分化得到不定芽。另外,针对苦瓜花药培养易于产生愈伤组织而难以再生不定芽的问题,首次对苦瓜花药和愈伤组织的内源激素含量进行了研究。主要的研究结果如下：(1)苦瓜小孢子发育时期与花蕾长度,花药颜色密切相关。应当以花蕾长度和花药颜色双重标准衡量小孢子的发育时期。小孢子处于单核后期时,绿脆纵横径最小,纵径为2.597 mm,横径为1.933 mm；其他三个品种花蕾纵径在4.024-4.213 mm,横径在1.998-2.050 mm。花药颜色为青白至微黄也是小孢子发育到单核后期的特征。(2)4℃低温预处理对苦瓜花药愈伤组织诱导率的影响因品种而异,经冷处理后,长白诱导率下降,碧秀、大白和胖妞的诱导率都显著提高。总体而言,苦瓜花药冷处理时间不宜过长,超过4天会显著降低诱导率,其最适冷处理时间为1-2天。(3)当MS培养基中2,4-D浓度为0.5 mg/L,6-BA浓度2.0 mg/L时,各品种的花药愈伤组织诱导率最高,其中碧秀苦瓜达到了79.42%。2,4-D浓度的变化对花药愈伤组织的诱导影响明显,低浓度的2,4-D(0.1 mg/L)与BA各水平组合,都不能很好地让苦瓜花药长出愈伤组织,2,4-D浓度过高又会造成愈伤组织膨大迅速,褐化死亡也加快。(4)苦瓜花药愈伤组织的诱导率随蔗糖浓度升高而显著降低,较高浓度的蔗糖可以有效抑制花药壁等体细胞发生愈伤组织,为了保证花药诱导率的同时也保证愈伤组织来源,培养基中添加50 g/L蔗糖最为适宜。(5)培养温度和光周期对苦瓜花药愈伤组织诱导有着重要影响。黑暗条件下比较容易产生愈伤组织,但是愈伤组织的质地很疏松,颜色发白；光照条件下产生愈伤组织比较慢,但质地致密,颜色绿。温度越高,花药愈伤组织诱导率越高,但质地更为疏松。25℃暗培养1周再转至光培养得到的诱导率最理想。(6)诱导阶段得到的愈伤组织按照外观和质地大致可分为两类：一类为黄白色、疏松状愈伤组织；另一类呈黄绿色、光滑颗粒状。(7)在TDZ 0.5 mg/L和2,4-D 0.1 mg/L的组合中添加三十烷醇2.0 mg/L,碧秀苦瓜部分黄绿色愈伤组织出现深绿色芽点并最终形成不定芽,这是首次在苦瓜花药培养中分化出不定芽。在激素组合为谷氨酰胺0.1 mg/L+TDZ 0.5 mg/L+2,4-D 0.1 mg/L的培养基中,黄白色愈伤组织出现3 cm左右的竖状突起。在激素组合6-BA 0.5 mg/L+NAA 1.0 mg/L中,长白苦瓜的部分黄绿色愈伤组织分化出了根。(8)在本试验中,长白苦瓜分化出不定根33条,根尖细胞染色体观察表明,其中4条为单倍体根,单倍体分化率为12.12%,其余为混倍体,其中三倍体较多。(9)应用高效液相色谱法(HPLC)研究了苦瓜外植体和愈伤组织内源激素含量,结果表明：苦瓜易于诱导愈伤组织与其内源IAA含量较高关系密切；高含量ZT对于不定芽的分化非常重要；高含量的GA3会抑制不定芽的发生,低水平的GA3/ZT值对于不定芽分化至关重要。更多还原

【Abstract】 Bitter melon (Momordica charantia L.) is not only a valuable vegetable crop but also an important medicinal plant in China and East Asia. Anther culture is one of the most effective ways of obtaining haploid and homozygotic plants, and it has special significance for the genetic improvement of plants. In this study, four cultivars of bitter melon were used for anther culture, and it was first time to differentiate buds in anther culture of bitter melon. Because explants of bitter melon were apt to form callus rather than adventitious buds, the present investigation measured and correlated endogenous hormone status on explants and calluses with their competence. The main results were as follows.(1) The relationship between microspore developmental stages and length of flower buds, color of anthers was established. When the microspores developed at the late-uninucleate stage, the flower buds were 2.597-4.213 mm in longitudinal diameter, 1.933-2.050 mm in diameter and the anthers were green-white or light green.(2) After 4℃cold pretreatment, callus induction rate of 3 genotypes increased while Changbai decreased. Cold pretreatment to anthers for 1-2 days resulted in the highest callus formation rate. While anthers pretreated more than 4 days, callus could hardly be induced.(3) The callus formation rate of bitter melon reached the highest when medium added with 2,4-D 0.5mg/L and BA 2.0 mg/L, and Bixiu reached 79.42%. Low concentration of 2, 4-D (0.1 mg/L) in combination with each level of 6-BA was unable to induce callus. However, with the concentration of 2,4-D up to 1.0 mg/L, the callus was prone to brown.(4) The induction rate decreased when the concentration of sucrose increased. Higher concentration of sucrose could inhibited callus derived from anther wall.5% sucrose in medium caused a relatively higher induction rate.(5) Anthers cultured under illumination condition resulted in lower induction rate than those cultured in darkness, however callus under light were denser and more vigorous. The anthers cultured at 25℃in the dark for 7 days and then cultured under light resulted in the most high induction rate.(6) Callus could be morphologically divided into two types:one type was slight yellow, loosened callus. The other type was viridescence, granular, smooth callus.(7) On MS medium supplemented with TDZ 0.5 mg/L,2,4-D 0.1 mg/L and triacontanol 2.0 mg/L, adventitious buds differentiated from callus of Bixiu. On MS medium supplemented with glutamine 0.1 mg/L, TDZ 0.5 mg/L and 2,4-D 0.1 mg/L, protuberant structures were produced on the surface of callus. Callus of Changbai differentiated roots when medium supplemented with 6-BA 0.5 mg/L and NAA 1.0 mg/L.(8) Anthers of Changbai differentiated 33 roots, and chromosomal examination of root tip cells showed that 4 roots were haploids.(9) The endogenous hormone concentrations of the initial explants and calluses were determined by means of high pressure liquid chromatography (HPLC). The endogenous IAA was higher in the explants that were easier to induce callus, and higher concentrations of ZT was positive to buds differentiation, while GA3 was negative to the buds formation.更多还原