Factors Affecting Seed Germination and Establishment of an Efficient Germination Method in Sugar Pine (Pinus lambertiana Dougl.)

Abstract

Mature sugar pine (Pinus lambertiana Dougl.) trees produce large amounts of viable seeds but have seed dormancy. In this study, we used three sugar pine genotypes, 8877, 9306, and 9375, to test seed germination response. Seed germination from local sources varied greatly, and germination percentages were poor. There was a large variation in seed size and seed weight among the genotypes. Seeds of 9375 and 9306 were significantly larger and heavier (30.7 and 28.8 g/100 seeds, respectively) than 8877 (23.6 g/ 100 seeds). Three types of seeds—intact seeds, hulled seeds, and naked embryos—were examined for germination. Intact seeds failed to germinate due to the physical restraint and water impermeability of the seed. Chemical scarification with 5 M hydrochloric acid and 5 M sodium hydroxide did not soften the hard seedcoat and also failed to induce any germination of intact seeds. Hulled seeds resulted in an extremely low germination percentage (£5%) with abnormal seedling development even though the endosperm was water permeable. Germination of the hulled seeds was not increased by adding 1 mg·L gibberellic acid to the culture medium. Artificial opening of the hulled seeds created by longitudinal or horizontal cuts on the endosperm after removal of the seedcoat to avoid physical restraint and allow air exchange also failed to improve germination, indicating that inhibitors related to germination were present in the endosperm. However, naked embryos of all three genotypes germinated rapidly and uniformly with 70% to 95% germination percentage regardless of cold stratification treatment. Our data indicate that sugar pine seeds from the current source did not have physiological dormancy of embryos themselves, but dormancy was imposed by the seedcoat and endosperm. Using the naked embryos as donor explants, we have successfully established an efficient in vitro culture system. The protocol described here can be applied for the tissue culture and genetic transformation of sugar pine. Sugar pine (Pinus lambertiana Dougl.), a gymnosperm belonging to the family of Pinaceae prized for its economic and ecological value, is one of the most valuable softwood forest plant species in thewesternUnited States. Native to the region from northern Oregon to Baja California, it is the largest species in the genus and is the tallest and most massive pine tree (Ahlstrom, 1992; Cermak, 1992; Kinloch and Scheuner, 1990; Maloney et al., 2011). Sugar pine is the most susceptible to white pine blister rust (WPBR) caused by Cronartium ribicola. The disease is rated as one of the worst pandemics in history, and its impact on the sugar pine natural population has been devastating (Devey et al., 1995; Ferrell and Scharpf, 1992). Traditional breeding efforts to create rust-resistant sugar pine by hybridizing sugar pine with rust-resistant white pine species have been hindered by long breeding cycles, the availability of rust-resistant species, incompatibility barriers among species, and poor hybrid seed production (Fernando et al., 2005). Recent advances in genetic engineering have provided an alternative opportunity to generate resistant sugar pine varieties in a greatly shortened time frame (Malabadi and Nataraja, 2017; Maleki et al., 2018; Marti and Dodd, 2018). Because sugar pine tree does not sprout (Kinloch and Scheuner, 1990), it is very difficult to establish in vitro from vegetative parts of mother plants. Reproduction through seeds in vitro is the only option to explore. Seed propagation has several advantages for mass production in sugar pine. Sugar pine bears the longest cones of all conifers, so seeds are abundant (Cermak, 1992). Using seeds to initiate in vitro culture normally results in less contamination than using vegetative parts of plants. However, sugar pine seeds are difficult to germinate and are characterized by irregular germination from diverse sources (Baron, 1978; Krugman, 1966). Seed germination is a complex process governed by internal and external factors. Among factors affecting germination, the state of the seeds themselves is the most important. Some seeds might be dormant while others are not (Bewley, 1997; Nelson, 2015). It is commonly believed that tree seeds possess some types of dormancy, physically or physiologically (or both). Studies on the dormancy and germination in Pinus species were reported, and different factors, alone or in combination, were attributed to dormancy in different Pinus species. Researchers found that seedcoat played an important role in seed dormancy, and germination could be improved by removal of seedcoat (Barnett, 1972, 1976), whereas others discovered that inhibitors existed in seedcoat and endosperm were related to seed dormancy (Li et al., 1989; Xin, 2008). Studies noted that dormancy was caused by underdeveloped embryos, and seedcoat removal alone could not overcome dormancy; thus, special treatment such as cold stratification was needed for germination (Carpita et al., 1983; Dong et al., 2002; Stone, 1957). Among treatments to break dormancy and thus improve germination, cold stratification is the most widely used. Various studies have shown its stimulating effect on seed germination in different Pinus species (Barnett, 1997; Cooke et al., 2002; Donald, 1987; Ghildiyal et al., 2009; McLemore and Czabator, 1961). On the other hand, some studies found that stratification did not affect seed germination (Nelson, 2015; Tanaka, 1984). Stratification temperature and duration exerted a significant effect on germination (Allen 1960; Malik and Shamet, 2008; Malik et al., 2008). The inductive effect of stratification was shown to vary among seed sources (Schubert, 1955; Skordilis and Thanos, 1995). Another commonly used treatment to promote seed germination is the exogenous application of gibberellic acid (GA3). It is well known that the plant hormone GA3 has a function in overcoming dormancy and stimulates seed germination in some species (Kucera et al., 2005). Studies with certain Pinus species supported this claim (Kumar et al., 2014; Lavania et al., 2006; Zhao and Jiang, 2014). Chemical scarification is often used to soften hard seedcoat or to rupture seedcoat to increase germination (Pitel and Wang, 1989). Few studies have been done in sugar pine on seed dormancy and germination. In our preliminary experiments, no germination could be obtained with intact seeds. In the present study, we investigated factors governing dormancy and germination in sugar pine. Ultimately, the objective of this study was to develop a reliable and rapid in vitro germination protocol in sugar pine. To achieve this goal, we sought to 1) examine Received for publication 2 Nov. 2020. Accepted for publication 8 Dec. 2020. Published online 15 February 2021. We thank Richard S. Dodd and Angel Fernandez i Marti from the Department of Environmental Science at University of California–Berkeley for providing seeds used in this study. This work was funded by the Innovative Genomics Institute of the University of California-Berkeley. M.-J.C. is the corresponding author. E-mail: [email protected]. This is an open access article distributed under the CC BY-NC-ND license (https://creativecommons. org/licenses/by-nc-nd/4.0/). HORTSCIENCE VOL. 56(3) MARCH 2021 299 seed morphology and structure of sugar pine varieties; 2) determine germinability of three types of seeds (intact seeds, hulled seeds, and naked embryos); 3) investigate the effects of tissues surrounding embryos on seed dormancy and germination; and 4) determine the effectiveness of cold stratification, chemical scarification, GA3 treatment, and artificial opening on seed germination. The results of this study can be applied to establish a tissue culture and genetic transformation protocol for sugar pine. Materials and Methods Seed source. Three genotypes of mature sugar pine seeds, 8877 (source #322.40), 9306 (source #741.40), and 9375 (source #534.65), were obtained from the LA Moran Reforestation Center located in Davis of California (Fig. 1A). Half of the seeds were stored at a room temperature of 22 C. The remaining half were put in a small box and kept at 4 C in a cold room for 3 months for cold stratification. Seed structure and morphological characteristics. The weights of three samples of 100 randomly selected seeds from each variety were measured. Seed structure and morphology were examined by sequential removal of tissues of seed components. On the basis of the examination of seed structure, three types of seeds were created with sequential removal of tissues surrounding the embryo: 1) intact seeds (including outer hard seedcoat, inner papery seedcoat, endosperm, and embryo) (Fig. 1A); 2) hulled seeds (containing endosperm and embryo without seedcoat) (Fig. 2A); 3) naked embryos (embryo only without seedcoat and endosperm) (Fig. 2B). These seeds were germinated with different treatments to investigate the effects of various seed components on germination. General sterilization procedure. In preliminary experiments, we experienced high contamination rates when culturing seeds in vitro. The following sterilization procedure worked best for reducing the contamination rate. Intact seeds: intact seeds were placed under running water for 4 h, then immersed in 30% germicidal ultra bleach (Pure Bright, 8.25% sodium hypochlorite) with the addition of 2 drops of Tween-20 for 1 h. Sterilized seeds were rinsed three times, 5 min each, with sterile water, then soaked in sterile water for 24 h. Hulled seeds: intact seeds were carefully cracked with a pair of pliers. The outer hard seedcoat was removed, then the inner papery seedcoat was also removed with pointed tweezers. Hulled seeds were immersed in 30% germicidal ultra bleach for 20 min. Sterilized hulled seeds were rinsed three times, 5 min each, with sterile water, then soaked in sterile water for 24 h. Naked embryos: the sterilization procedure was the same as for hulled seeds. After 24 h soaking in