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