Kiełkowska Agnieszka, Adamus Adela, Baranski Rafal
Institute of Plant Biology and Biotechnology, Faculty of Horticulture, University of Agriculture in Krakow, Al. 29-Listopada 54, 31-425 Krakow, Poland.
In Vitro Cell Dev Biol Plant. 2014;50(3):376-383. doi: 10.1007/s11627-014-9597-1. Epub 2014 Mar 8.
In this work, we describe an improved protocol for induced parthenogenesis and ovule culture of carrot ( L.). The effects of pollination with parsley pollen and/or 2,4-dichlorophenoxyacetic acid (2,4-D) treatment on the stimulation of parthenogenesis were studied using heterozygous donor plants of 30 varieties and breeding populations of carrots. Isolated ovules, cultured , enlarged and developed embryos or calli. The application of 2,4-D on pollinated flowers stimulated callus development but did not increase the frequency of embryo development from ovules and, thus, was not useful for increasing the frequency of haploid plant recovery. The efficiency of embryo development was accession-dependent and varied from 0 to 24.29%. In optimized conditions, most accessions responded by embryo development exclusively. The highest frequency of embryo development was observed from ovules excised from ovaries 20-22 d after pollination with parsley pollen. Among several media used for ovule culture, 1/2-strength Murashige and Skoog medium with 0.06 μM indole-3-acetic acid (IAA) was the best. It allowed the production of embryos at a similar frequency as on the media supplemented with kinetin, gibberellic acid, putrescine, or thidiazuron, but restricted callus development. Most plants obtained were haploids and diploids derived from parthenogenesis, as evidenced by homozygosity at three independent loci based on isozyme and PCR analyses. In total, considering haploids and embryo-derived homozygous diploids together, 72.6% of regenerated plants were of gametic origin.
在本研究中，我们描述了一种改良的胡萝卜（L.）孤雌生殖诱导及胚珠培养方案。使用30个品种的杂合供体植株和胡萝卜育种群体，研究了用欧芹花粉授粉和/或2,4 - 二氯苯氧乙酸（2,4 - D）处理对孤雌生殖刺激的影响。分离的胚珠经培养后，胚胎或愈伤组织得以扩大和发育。在授粉花朵上施用2,4 - D刺激了愈伤组织的发育，但并未增加胚珠胚胎发育的频率，因此，对提高单倍体植株获得频率并无帮助。胚胎发育效率因材料而异，范围为0至24.29%。在优化条件下，大多数材料仅通过胚胎发育做出反应。在用欧芹花粉授粉后20 - 22天从子房切下的胚珠中观察到最高的胚胎发育频率。在用于胚珠培养的几种培养基中，添加0.06 μM吲哚 - 3 - 乙酸（IAA）的1/2强度Murashige和Skoog培养基效果最佳。它能以与添加激动素、赤霉素、腐胺或噻二唑的培养基相似的频率产生胚胎，但限制了愈伤组织的发育。基于同工酶和PCR分析，在三个独立位点的纯合性证明，获得的大多数植株是孤雌生殖产生的单倍体和二倍体。总体而言，将单倍体和胚源纯合二倍体合计，72.6%的再生植株起源于配子。
