Prem Deepak, Solís María-Teresa, Bárány Ivett, Rodríguez-Sanz Héctor, Risueño María C, Testillano Pilar S
Plant Development and Nuclear Architecture, Centro de Investigaciones Biológicas, CIB-CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain.
BMC Plant Biol. 2012 Aug 2;12:127. doi: 10.1186/1471-2229-12-127.
Microspore embryogenesis represents a unique system of single cell reprogramming in plants wherein a highly specialized cell, the microspore, by specific stress treatment, switches its fate towards an embryogenesis pathway. In Brassica napus, a model species for this phenomenon, incubation of isolated microspores at 32°C is considered to be a pre-requisite for embryogenesis induction.
We have developed a new in vitro system at lower temperature (18°C) to efficiently induce microspore embryogenesis throughout two different developmental pathways: one involving the formation of suspensor-like structures (52.4%) and another producing multicellular embryos without suspensor (13.1%); additionally, a small proportion of non-responsive microspores followed a gametophytic-like development (34.4%) leading to mature pollen. The suspensor-like pathway followed at 18°C involved the establishment of asymmetric identities from the first microspore division and an early polarity leading to different cell fates, suspensor and embryo development, which were formed by cells with different organizations and endogenous auxin distribution, similar to zygotic embryogenesis. In addition, a new strategy for germination of microspore derived embryos was developed for achieving more than 90% conversion of embryos to plantlets, with a predominance of spontaneous doubled haploids plants.
The present work reveals a novel mechanism for efficient microspore embryogenesis induction in B. napus using continuous low temperature treatment. Results indicated that low temperature applied for longer periods favours an embryogenesis pathway whose first division originates asymmetric cell identities, early polarity establishment and the formation of suspensor-like structures, mimicking zygotic embryogenesis. This new in vitro system provides a convenient tool to analyze in situ the mechanisms underlying different developmental pathways during the microspore reprogramming, breaking or not the cellular symmetry, the establishment of polarity and the developmental embryo patterning, which further produce mature embryos and plants.
小孢子胚胎发生代表了植物中一种独特的单细胞重编程系统，其中高度特化的细胞——小孢子，通过特定的胁迫处理，将其命运转向胚胎发生途径。在这种现象的模式物种甘蓝型油菜中，将分离的小孢子在32°C下培养被认为是诱导胚胎发生的先决条件。
我们开发了一种新的低温（18°C）体外系统，以通过两种不同的发育途径有效诱导小孢子胚胎发生：一种途径涉及形成类胚柄结构（52.4%），另一种途径产生无胚柄的多细胞胚胎（13.1%）；此外，一小部分无反应的小孢子遵循类配子体发育途径（34.4%），发育为成熟花粉。18°C下遵循的类胚柄途径涉及从第一次小孢子分裂开始建立不对称身份以及早期极性，从而导致不同的细胞命运、胚柄和胚胎发育，这些由具有不同组织和内源性生长素分布的细胞形成，类似于合子胚胎发生。此外，还开发了一种新的小孢子衍生胚胎萌发策略，以使超过90%的胚胎转化为植株，其中自发加倍单倍体植株占主导。
本研究揭示了一种利用连续低温处理在甘蓝型油菜中高效诱导小孢子胚胎发生的新机制。结果表明，长时间施加低温有利于一种胚胎发生途径，其第一次分裂产生不对称的细胞身份、早期极性建立以及类胚柄结构的形成，类似于合子胚胎发生。这种新的体外系统提供了一个方便的工具，用于原位分析小孢子重编程过程中不同发育途径的潜在机制，包括细胞对称性的打破与否、极性的建立以及胚胎发育模式的形成，进而产生成熟胚胎和植株。
