Water and nitrogen stresses are major constraints for agricultural and forest productivity. Although the effects of water scarcity or nitrogen stress on plant growth, physiology, and yield have been widely studied, few studies have assessed the combined effects of both stresses. In the present study, we investigated the effects of different nitrogen forms (NO₃⁻N, NH₄⁺-N, and a combination of NO₃⁻N + NH₄⁺-N) on antioxidant enzyme activity, osmotic regulatory substances, and nitrogen assimilation in Chinese fir (Cunninghamia lanceolata) plantlets under drought stress (induced by 10% polyethylene glycol). We found that different N ionic forms had different effects on drought-stressed plantlets. Nitrogen supply greatly increased the activities of superoxide dismutase (SOD), peroxidase (POD) and polyphenol oxidase (PPO) when plantlets were exposed to water stress. The malondialdehyde (MDA) contents significantly decreased under the NH₄⁺ + water stress treatment. The proline (Pr) contents significantly increased in both the NO₃⁻N and NH₄⁺-N + water stress treatment. The nitrate reductase (NR) increased by 7.1% in the NO₃⁻ + water stress treatment, and the glutamine synthetase (GS), and the glutamate synthase (GOGAT) activity increased in all the nitrogen + water stress treatments. These results suggested that nitrogen supply could alleviate the adverse effects of drought stress on plants by enhancing antioxidant defense and improving nitrogen assimilation, while the effects on plant tolerance to drought stress varied with nitrogen ionic forms.

In vitro pollen germination provides a novel approach and strategy to accelerate genetic improvement of tree breeding. Studies about pollen germination and tube growth of Chinese fir are limited. Therefore, this study aimed to investigate the effects of sucrose, boric acid, pH, and time of incubation on pollen germination and tube growth. Pollen from 9 clones were selected. In vitro germination was performed in basic media as control, and in different concentrations of sucrose (0, 10 and 15%), boric acid (0.01, 0.1 and 0.2%), and pH levels (4.5, 5 and 7). Pollen germination rates and tube growth were recorded periodically at 1, 12, 24, and 48 h. The results showed that sucrose imposes significant effects on pollen germination and tube growth. The effects are most obvious at concentration of 15%. Boric acid significantly promoted germination and tube growth. The promotion was most notable in lower concentration of 0.01%. The media adjusted to pH 7.0 boosted the germination and pollen tube growth. The optimum time of incubation was 24 and 48 h for pollen germination and tube growth, respectively. Sucrose, pH, and time of incubation were positively correlated, whereas boric acid negatively correlated with pollen germination and tube growth. This study provided experimental evidences for selecting viable pollens for Chinese fir breeding.

RNA isolation with RNA in a high quantity is a basic analytical method in plant genetics, molecular biology and related physiological investigations. To understand the genetic and molecular biology of Chinese fir, sufficient high-quality total RNA must be obtained for cDNA library construction and other downstream molecular applications. However, extracting RNA from Chinese fir is difficult and often requires the modification of existing protocols. Chinese fir tissues containing large amounts of polysaccharides and polyphenol compounds and are one of the most difficult plant tissues for RNA isolation. Therefore, we developed a simple method for extracting high-quality RNA from Chinese fir tissues. RNA isolations were performed within two hours, RNA quality was measured for yield and purity. Total RNA obtained from this procedure was successfully used for cDNA library construction, RT-PCR and transcriptome sequencing. It was proven that extracted RNA was intact and suitable for downstream molecular applications, including RT-PCR and qPCR, and other downstream molecular applications. Thus, this protocol represents a simple, efficient, and low-cost method.

Pollen grains produce certain metabolites, which can improve or inhibit germination and tube growth. Metabolomic analysis of germinating and growing Chinese fir pollen has not been reported. Therefore, this study aimed to analyse metabolites changes, content and expression in the germinating pollen of Chinese fir. To understand the metabolic differences, two clones from Chinese fir were selected. Metabolomics analyses were performed on three stages (1-, 24- and 48-h) during in vitro pollen germination. The metabolites profiles at different time points were analyzed by using liquid chromatography-mass spectrometry. The results showed that 171 peaks were screened; the corresponding differential metabolites of 121 peaks were classified into nine types of substances. The expression of metabolites showed significant differences across and between clones, and the variation was evident at all germination stages. The expression was obvious at the early stage of germination, which differed clearly from that of the late stage after pollen tube growth. Moreover, the metabolites were mainly enriched in 14 metabolic pathways. Pollen germination and tube growth and metabolites expressions changed per incubation time. Since this work is preliminary, we suggest further investigations to understand the relationship between the differential metabolites and pollen development, and factors affecting pollen germination process.

In order to ascertain the optimal hormone combination for Chinese fir (Cunninghamia lanceolata) leaf cuttings, an L₁₆(4)⁴ orthogonal test of 6-benzylaminopurine (6-BA), 1-naphthaleneacetic acid (NAA), and indole-3-butyric acid (IBA) (0, 10, 30, or 50 mg · L⁻¹ of each exogenous hormone) immersion for 5, 10, 15, or 20 min was conducted. Callus initiation rate and rooting promotion rate were mainly affected by treatment time, root length increase by 6-BA concentration, and bud germination rate and plantlet formation rate by NAA concentration. The expected optimal combination for callus initiation rate was 50 mg · L⁻¹ 6-BA + 0 mg · L⁻¹ NAA + 30 mg · L⁻¹ IBA + 10 min; for rooting promotion rate, it was 0–10 mg · L⁻¹ 6-BA + 10 mg · L⁻¹ NAA + 30 mg · L⁻¹ IBA + 20 min; for bud germination rate, it was 50 mg · L⁻¹ 6-BA + 0–10 mg · L⁻¹ NAA + 0–30 mg · L⁻¹ IBA + 20 min; and for seedling formation rate for No. 4, it was 10 mg · L⁻¹ 6-BA + 10 mg · L⁻¹ NAA + 0 mg · L⁻¹ IBA + 20 min. Light microscopy image analysis revealed that a cluster of primordial cells was produced 40 days after cutting, and mastoid cells developed into peninsula cells in calli that were cultured for 50 days.