Multiple Methods Synergistically Promote the Synchronization of Somatic Embryogenesis Through Suspension Culture in the New Hybrid Between Pinus elliottii and Pinus caribaea

第一作者：Fengqing Li

第一单位：南京林业大学

通讯作者：Jisen Shi

 Abstarct 

背景回顾：Pinus elliottii × Pinus caribaea is an interspecific pine hybrid of major economic importance. Somatic embryogenesis and plant regeneration in P. elliottii × P. caribaea on solid medium have been reported previously; however, a current limitation is the lack of a stable and effective method for its commercial use. 

湿地松×加勒比松是一种具有重要经济价值的种间杂交松。前人已研究并报道了在固体培养基上湿地松×加勒比松的体细胞胚胎发生和植株再生；然而，目前，还未建立可用于规模化繁殖的有效方法。

主要研究：The objective of this study was to establish a suspension culture system and evaluate the effect of multiple methods synergistically on the synchronization of embryo development in P. elliottii × P. caribaea. 

本研究的目的是建立一个悬浮培养体系，并探讨了多种方法协同作用对湿地松×加勒比松的体细胞胚胎发生同步化的影响。

结果1-胚性愈伤组织悬浮培养体系建立：For the former, a protocol to initiate and establish a suspension culture system of P. elliottii × P. caribaea was presented. Based on biomass growth, the growth of embryogenic calli (EC) followed an S-shape curve in suspensions grown for a 15-day period, and the exponential phase of cell suspensions was reached between days 3 and 6. The initial packed cell volume (PCV) and revolutions per minute (rpm) have a significant effect on the proliferation of EC, and the highest proliferation multiple reached 6.86 (±0.06) at the initial density of 5 ml PCV under a 9–10 days transfer interval in the dark on a rotary shaker at 70 rpm. 

基于愈伤组织生物量生长的基础上，胚性愈伤组织在15d的培养周期内呈S型曲线，在继代培养后的3-6d，细胞悬浮液的生长达到指数阶段。初始的密实细胞体积和转速对胚性愈伤的增殖有显著影响，在初始密实细胞体积为5mL，转速为70 rpm，且在暗培养条件下，悬浮液的继代周期为9-10 d 为佳，最高增殖倍数达到6.86（±0.06）。

结果2-ABA、N源的种类和浓度、低温和PEG对体细胞胚胎同步发生影响显著：For the latter, the influence of abscisic acid (ABA), ammonium (NH4+), nitrate (NO3--), low temperature, and polyethylene glycol (PEG) on somatic embryogenesis was very significant. When EC were suspended in the medium at a presence of 37.84 μM/L ABA, as many as 274 mature cotyledonary embryos/ml PCV of cells were thereafter formed in the mature medium, and 266 somatic embryos were obtained on mature medium after suspension culture in liquid medium containing 10 mmol/L NH4+ and 30 mmol/L NO3-. Furthermore, reducing the concentration of 2,4-dichlorophenoxyacetic acid gradually and at 4°C incubation for 12 h in the initial exponential phase could promote the synchronization of somatic embryogenesis, which resulted in 260 mature cotyledonary embryos. 

当胚性愈伤在含有37.84 uM/L ABA的培养基中悬浮培养后，在成熟培养基上培养45d后，每mL密实细胞体积胚性细胞可形成多达274个成熟子叶胚；在含有10 mmol/L NH4+ 和30 mmol/L NO3- 的液体培养基中悬浮培养后，在成熟培养基上可获得266个成熟体细胞胚；在悬浮培养过程中逐步降低2,4-D浓度，并在每个生长周期的指数生长期在4℃下培养12h，亦可促进体细胞胚胎发生的同步化，可产生260个成熟子叶胚。

结论：This suspension culture system and method of synchronic control can be used in the large-scale production of P. elliottii× P. caribaea seedlings.

本研究建立的悬浮培养体系和同步化控制方法可用于湿地松×加勒比松体胚苗的规模化生产。

doi: https://doi.org/10.3389/fpls.2022.857972

Journal: Frontiers in Plant Science

Published date: April 25, 2022