两个花椰菜品种再生体系的研究

以2个花椰菜品种的子叶和上胚轴为外植体,以MS为基本培养基,设置不同激素浓度,建立再生体系。结果表明:子叶作为外植体时不定芽分化率低,子叶大部分发生黄化。上胚轴作为外植体不定芽分化率较高,在适合的培养基上均能达到100%,‘祁连白雪’上胚轴最佳诱导培养基为MS+6-BA 1.0 mg/L+IAA 0.5 mg/L,‘新东海明珠80天’的最佳诱导培养基为MS+6-BA 1.5 mg/L+IAA0.5 mg/L。分化后得到的不定芽在MS+6-BA 0.5 mg/L培养基上伸长后,在MS+IAA 0.5 mg/L的培养基上生根,生根率达100%,移植成活率95%。

切除上胚轴对绿豆子叶衰老逆转及其核酸和蛋白质代谢的效应

在绿豆子叶衰老达到不归点(萌发后5～6d)前切除上胚轴可使开始衰老的子叶中核酸和蛋白质含量回升,衰老短期逆转。衰老不归点后的子叶中核酸和蛋白质变化的主要特征是:丧失了较多的核主带DNA、25S、18S rRNA以及大部分可溶性蛋白质组分,一种小分子DNA 组分完全消失。不归点前切除上胚轴可使上述核酸和蛋白质组分明显增加,表明子叶衰老的逆转可能与这些重要功能物质的回升有关。在切除上胚轴的茎顶涂抹IAA,能阻止子叶核酸和蛋白质回升,也消除了切除上胚轴对子叶裹老的逆转作用。

花椰菜再生体系的优化

以子叶和上胚轴为外植体,研究了不同激素浓度下花椰菜离体培养的再生体系。结果表明,上胚轴作为外植体时不定芽分化率较高,在不加IAA或添加0.5 mg/L IAA的培养基上均能达到100%,最佳诱导培养基为MS+1.0 mg/L 6-BA+0.5 mg/L IAA。子叶作为外植体时不定芽分化率低,最高为40%。

红江橙与柠檬愈伤组织诱导的研究

以MT为基本培养基,采用L9(34)正交设计法,研究了不同培养基配方对红江橙和柠檬不同外植体愈伤组织诱导的影响。结果表明,上胚轴、下胚轴、子叶均能诱导出愈伤组织,但不同外植体在形成愈伤组织的时间和过程上有差别;红江橙上胚轴、下胚轴、子叶愈伤组织诱导的最佳实验培养基配方分别为MT+2,4-D 1.00 mg/L+6-BA0.50 mg/L+蔗糖30 g/L、MT+2,4-D0.50 mg/L+6-BA0.50 mg/L+蔗糖40 g/L、MT+2,4-D 0.50mg/L+6-BA1.00 mg/L+蔗糖20 g/L,此时愈伤组织诱导率均为100%;柠檬上胚轴、下胚轴愈伤组织诱导的最佳实验培养基配方分别为MT+2,4-D1.00 mg/L+6-BA0.50 mg/L+蔗糖30 g/L、MT+2,4-D1.00 mg/L+6-BA2.00 mg/L+蔗糖20 g/L,此时愈伤组织诱导率分别为55%、29%。

荞麦愈伤组织的高频率诱导和植株再生研究

研究了高频率诱导荞麦愈伤组织和再生植株的组培技术。在附加 0 .5 mg/L 2 .4-D和 KT的 MS4培养基上 ,胚轴和子叶外植体愈伤组织的诱导率高达 81.3 %～ 91.2 % ,胚轴不定芽的诱导率为 3 4 .5 %～ 41.2 %。将胚轴在附加 0 .5 mg/L 2 .4—D的 MS2上预培养 15 d左右再转到 MS4上 ,不定芽诱导率可达 5 3 .3 %。不定芽也可从培养于 MS5 (附加 0 .5 mg/L KT)中的外植体上直接产生。当不定芽转到附加 0 .5 mg/L NAA的 MS7培养基上可诱导生根。

油松成熟胚子叶-胚轴段的离体培养与不定芽诱导

以油松成熟胚的子叶-胚轴段为起始外植体,进行离体培养诱导不定芽,以不同浓度的活性炭对油松不定芽进行继代培养。结果表明:子叶-胚轴段可以诱导出不定芽;在1/2MS培养基上附加3 mg/L的6-BA和0.5 mg/L的NAA,外植体不定芽的诱导率达46.7%,平均增殖系数是10,最大增殖系数为24;1/2MS培养基中附加2 mg/L的活性炭对油松不定芽增殖和生长最有利。

‘蜂蜜罐’枣遗传转化条件的优化及Bt基因的导入

为建立‘蜂蜜罐’枣高效遗传转化体系,以‘蜂蜜罐’枣胚培苗上胚轴和子叶为外植体进行转化,研究了不同菌液浓度、侵染时间、共培养时间、AS(乙酰丁香酮)浓度和侵染方式对gus(β-葡糖醛酸糖苷酶)基因瞬时表达率的影响。结果表明,当菌液OD_(600)值为0.5,真空辅助侵染10 min,共培养3 d,上胚轴和子叶外植体的共培养培养基中分别添加20和10 mg·L^(-1)乙酰丁香酮时,其gus瞬时表达率均最高,为最佳遗传转化体系。利用已优化的遗传转化体系进行Bt基因转化,共获得24个PCR(聚合酶链式反应)检测呈阳性的再生芽;对PCR检测呈阳性的不定芽进行伸长培养和不定根诱导,最终获得完整转基因植株。本研究初步获得了转Bt(苏云金芽孢杆菌)基因的枣植株,为通过转基因手段进行枣抗虫新种质选育提供可能。

<i>Agrobacterium</i>-Mediated Transformation of Mexican Lime (<i>Citrus aurantifolia</i>Swingle) Using Optimized Systems for Epicotyls and Cotyledons

Transgenic Mexican lime (Citrus aurantifolia Swingle) was produced through two explant sources, each using systems previously optimized for each source. One used epicotyls segments, which was the predominant explant for transgenic Citrus production following co-cultivation with Agrobacterium, and has a well-established protocol. The other procedure used embryo cotyledons from mature seeds, which was developed in our lab as an alternative for stable Citrus transformation. Cotyledon transformation and regeneration protocols were optimized by comparing variables in culture medium composition on shoot regeneration and four parameters in transient transformation. The optimized protocols were compared, and frequency of regeneration, frequency of transgenic plant-recovery and stable transformation efficiency indicated the superiority of the cotyledon protocol for Agrobacterium-mediated genetic transformation in Mexican lime. The tissue choice resulted in marked improvement in shoot regeneration (14.1% of explants producing shoots in epicotyls;55.8% in cotyledons), stable transformation frequency (11.4% of epicotyls explants;40.2% in cotyledons), and frequency of transgenic plant-recovery (37.9% in epicotyl explants;92.6% in cotyledons). Thus, easy availability of explants using embryo cotyledons from mature seeds, technical simplicity, shortening of transformation time-course, and higher transformation and regeneration frequencies makes this new system an attractive alternative over the previously published Citrus transformation protocols. In the course of this project, we generated Mexican lime with a Recombinase Mediated Exchange Cassette landing pad, which was designed for stacking transgenes.