Introduction
Pinus thunbergii (an evergreen tree species) is resistant to sea mist and wind; thus, it is employed extensively in coastal urban hill greening and as a coastal windbreak in China, Japan, and Southern Korea (Han et al., 2008; Zhang et al., 2009; Mao et al., 2019). However, it is susceptible to pine wilt disease caused by the Bursaphelenchus xylophilus nematode, which has propagated worldwide. The epidemic of pine wilt disease has led to a massive decline of Pinus thunbergii trees, and threatened entirePinus ecosystems (Hussain et al., 2021). The deployment of PWN-resistant genotypes is an important strategy to control the disease (Li et al. 2022). Thus, a long-term breeding project was undertaken to select resistant clones for pine trees in Japan (Futai K and Furuno T, 1979; Endo R and Fukuhara K, 2017; Fujimoto et al., 1989). Somatic embryogenesis has proved to be one of the most promising techniques for the mass propagation of conifers. Our group had reported the somatic embryogenesis and plant regeneration of nematode-resistant Pinus thunbergii (Sun et al., 2019; Wu et al., 2008; Li et al., 2013; Zhu et al., 2019). Besides, many regenerated plants had been obtained fromPinus species via somatic embryogenesis, including Pinus pinea (Carneros et al., 2009), Pinus elliottii (Yang et al., 2020), Pinus thunbergii (Maruyama et al., 2005; 2012) andPinus radiata (Montalban et al., 2012). However, reports on the application in afforestation of SRPs were rare, where low survival rate was the primary challenges.
The transition from lab- to field-seedlings is a major obstacle in plant breed technology. The difference of microenvironment between seedlings cultured in lab and in field made the regeneration plant delicate and weakened root system which resulted in a survival rate (Zhou et al., 2022). Transplantation survival directly determines whether tissue-cultured regeneration plant can be utilized in field production. An effective acclimation cultivation project could generate high-quality seedlings to promote their survival rate (Matin et al., 2000). As a facilitator, sugar is essential for plant growth and mediating effective molecular signaling processes for plant development (Smeekens et al., 2010). During micropropagation, sucrose-cleaving enzymes could rapidly promote leaf growth. Moreover, the transport of sugars in the phloem influenced the development of aboveground organs and roots (Pantin et al. 2012; Rottmann et al. 2018; Wang et al. 2013). Therefore, the application of sugar in vitro was key toward enhancing the growth and development of plantlets (Rolland et al. 2006; Zhu et al. 2018). Culture medium and transplantation stroma provide essential nutrients, and possess good water retention, air permeability, and acid-base buffering capacities for plant regeneration. The selection of the transplantation stroma is one of the key factors determining the survival rate of transplants. The survival rate of P. densiflora plantlets was up to 60% in mixture of transplantation stroma containing vermiculite-perlite-sand (1:1:1) (Zhu et al., 2010). The was approximate. Brassinosteroids (BRs) have been extensively applied in plant growth studies, as it is considered to be an effective and ecofriendly phytohormone that regulates the differentiation of root epidermal cells and root hairs, while facilitating the formation of lateral roots (Wang et al., 2009; Nolan et al., 2020). Further, the beneficial effects of spectral treatments on plant growth have been widely observed (Ranade et al. 2016; Li et al. 2013). These factors justify the enormous research efforts that have been undertaken to improve nursery protocols for the generation of highly quality plantlets. However, these studies, particularly the application of BRs and spectral treatments for the regulation of plant growth, have been less frequent for pine tree species (Lazzarini et al. 2018). Previously, our group reported the plant regeneration of nematode-resistantPinus thunbergii through somatic embryogenesis (Sun et al., 2019). To further promote the application in afforestation of SRPs, we studied the growth factors of sucrose, medium types, BRs, and spectral treatments on the of nematode-resistant Pinus thunbergii SRPs. In addition, their survival rates were continuously monitored. The aim of this study was to develop a reliable acclimation cultivation project that would improve the quality of pinus species plantlet and increase the transplanted survival rate in field.
Materials and methods