Cryopreservation of mature zygotic embryos, shoot bud regeneration, and field establishment of Pinus elliottii var. elliottii x Pinus caribaea var. hondurensis in vitro-derived plants
Lilian P. E. Ayala, Claudia V. Luna, Elsa A. Brugnoli, Fabiana D. Espasandin, María J. Duarte, Ana M. González, María E. Gauchat, Paloma Moncaleán Guillén, Pedro A. Sansberro
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引用次数: 0
Key message
The developed protocol for organogenesis, in vitro plantlet production, and cryopreservation opens the possibility for mass propagation of hybrid pine (Pinus elliottii var. elliottii x Pinus caribaea var. hondurensis).
Abstract
The low seed production of the interspecific hybrid Pinus elliottii var. elliottii x Pinus caribaea var. hondurensis restricts its commercial expansion, making it necessary to ensure efficient cryopreservation and a propagation protocol with no genetic variability. Mature zygotic embryos (MZEs) were cultured in Murashige and Skoog (MS) medium containing 6-benzyladenine (BA) and thidiazuron (TDZ). After 45 days in culture, the highest rate of regeneration (86.7 ± 8.8%) and the maximum number of differentiated buds per responsive explant (15.5 ± 2.8) were achieved from explants cultivated on MS with BA and TDZ (0.5 μM each). Cryopreservation of zygotic embryos using a simple desiccation step and a direct immersion into liquid nitrogen did not affect regeneration and would enhance embryo storage duration. Half-strength MS enriched with sucrose (0.09 M) and gelled with gellan gum (4 g L− 1) under forced ventilation culture was used for shoot elongation. Subsequently, 73 ± 6.7% of shoots produced roots after pretreatment with 1.25 mM indole-3-butyric acid solution for 5 min and culture on quarter-strength MS with sucrose (0.045 M). The regenerated plantlets were successfully transferred to ex vitro conditions. The procedure took 160 days and comprised the adventitious bud formation from cryopreserved MZEs, shoot elongation, rooting, and plantlet acclimation. Considering that water deficit is the major strain during forest establishment, a controlled experiment was carried out to determine the competence of plantlets to overcome this stress. Next, field studies assessed the survival rate and growth of 16-month-old plants. Our results indicated that the field performance of tissue-culture-derived plants is similar to seedlings and rooted cutting plants. Additionally, inter-simple sequence repeat marker analysis revealed the genetic uniformity among the in vitro-raised plants, demonstrating the reliability and validity of the procedure. Thus, the developed regeneration and cryopreservation protocol for mature zygotic embryo explants is a valuable alternative for breeding programs and commercial P. elliottii x P. caribaea propagation.
湿地松(Pinus elliottii var. elliottii)和北美松(Pinus caribaea var. hondurenis)成熟合子胚的低温保存、芽再生及离体植物的田间建立
该研究为杂交松(Pinus elliottii var. elliottii x Pinus caribaea var. hondurensis)的大量繁殖提供了可能。摘要种间杂交种油松(Pinus elliottii var. elliottii)与油松(Pinus caribaea var. hondurensis)的种子产量低，限制了其商业推广，因此必须确保有效的低温保存和无遗传变异的繁殖方案。将成熟的合子胚胎(MZEs)培养在含有6-苄基腺嘌呤(BA)和硫代脲(TDZ)的Murashige和Skoog (MS)培养基中。培养45 d后，BA和TDZ(各0.5 μM)在MS上培养的外植体再生率最高(86.7±8.8%)，每个响应外植体分化芽数最多(15.5±2.8)。使用简单的干燥步骤和直接浸泡在液氮中冷冻保存的合子胚胎不影响再生，并且可以延长胚胎的保存时间。在强制通风培养条件下，添加蔗糖(0.09 M)和结冷胶(4 g L−1)的半强MS用于芽伸长。经1.25 mM吲哚-3-丁酸溶液预处理5 min后，在1 / 4强度MS (0.045 M)蔗糖培养基上培养，73±6.7%的再生苗成功转移到离体条件。该过程耗时160天，包括冷冻MZEs的不定芽形成，茎伸长，生根和植株驯化。考虑到水分亏缺是成林过程中的主要胁迫，通过对照试验确定了幼苗克服水分亏缺胁迫的能力。接下来，实地研究评估了16个月大的植株的存活率和生长情况。我们的研究结果表明，组培植物的田间表现与幼苗和有根扦插植物相似。此外，通过简单序列重复标记分析，揭示了该方法在离体栽培植株间的遗传一致性，证明了该方法的可靠性和有效性。因此，成熟的合子胚外植体再生和低温保存技术是一种有价值的育种方案和商业繁殖的替代方案。
期刊介绍：
Trees - Structure and Function publishes original articles on the physiology, biochemistry, functional anatomy, structure and ecology of trees and other woody plants. Also presented are articles concerned with pathology and technological problems, when they contribute to the basic understanding of structure and function of trees. In addition to original articles and short communications, the journal publishes reviews on selected topics concerning the structure and function of trees.
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