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−1 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.
Xiuli Shen, Guochen Yang, and Zhongge (Cindy) Lu
To overcome the limitations of traditional propagation, this research was initiated to develop an alternative means for efficient production of Alexandrian laurel (Danae racemosa L. Moench). An in vitro propagation protocol has been developed for Danae racemosa L. Moench using seeds as a source of material for culture initiation. Seedlings were produced after seeds were cultured for 3 month on MS () medium. Shoot multiplication occurred on MS medium with or without 6-benzylaminopurine (BAP) with 100% multiplication percentage. However, shoot number was significantly increased from an average of 2.8 to more than six with the addition of 5 or 25 μM BAP. Among two indole-3-butyric acid (IBA) treatments tested for rooting of seedlings, incorporation of 5 μM IBA in MS medium significantly increased rooting percentage to 86.4% compared with 71.2% without IBA. The greatest number of roots (three) was produced by 5-minute IBA pulse. However, both IBA treatments significantly reduced root length. The longest root (12.8 mm) was observed on MS medium without any IBA treatment and the shortest (6.1 mm) was produced by IBA pulse. In vitro-propagated plantlets grew well after transfer to a substrate of peat and pine bark (1:1) in the greenhouse. No morphological variation was observed.
Xiuli Shen, William S. Castle, and Frederick G. Gmitter Jr
Casuarina cunninghamiana Miq. is an introduced species to Florida that has potential as a windbreak plant to help manage canker in citrus groves; however, only Florida sources can be used for that purpose. Local sources of Casuarina are generally adequate seed producers, but germination percentages are frequently poor. Thus, the causes of low seed germination and methods to improve germination were investigated using C. cunninghamiana and a local hybrid (C. equisetifolia L. × C. glauca Sieb. ex Spreng.). Seeds of the hybrid were larger and heavier (88 mg/100 seeds) than those of C. cunninghamiana (mean wt. 67 mg/100 seeds). Shrunken, insect-damaged, and empty seeds, present in all unsorted seed lots, were responsible for poor seed germination of the four seed sources studied. Petroleum ether separation improved germination by dividing seeds into floaters and sinkers. The floater fraction consisted of 47.5% to 93% insect-damaged seeds compared with 9.0% to 43.5% among sinkers. More than 50% of the sinkers were filled seeds and less than 21% in floaters. No empty seeds were sinkers except for one source of C. cunninghamiana. In sorted hybrid seeds, petroleum ether separation eliminated a large proportion of ungerminable seeds (floaters) and seed germination among sinkers was faster with a higher germination percentage than floaters. Cumulative germination of hybrid seeds in a trial involving two temperatures was 23.0% for sunken seeds at 30 °C at the end of 8 weeks compared with 1% of unsorted seeds. Temperature had no significant effect on seed germination. The germination percentage of hybrid seeds with seedcoats removed was 91.0% in the first week of culture compared with only 1.2% in the first week and 12.6% seed germination at the end of 8 weeks' culture of intact seeds.
Xiuli Shen, William S. Castle, and Frederick G. Gmitter Jr
This study examined the effects of plant growth regulators, explant types, and their orientations on in vitro shoot proliferation of Casuarina cunninghamiana Miq. and also the subsequent rooting ability of shoots. Results showed that shoot proliferation occurred only in shoot tip explants cultured vertically on Murashige and Skoog (MS) medium supplemented with 2 or 4 μM thidiazuron (TDZ). Neither 6-benzylaminopurine alone nor in a combination with 1-naphthalene acetic acid (NAA) or gibberellic acid had any effect on shoot proliferation. TDZ at 4 μM resulted in the greatest percentage of axillary bud sprouting (70%) and mean number of sprouts per explant (2.3). Additionally, no shoot proliferation was observed from detipped or single-node explants or from horizontally placed shoot tip explants when cultured on the same TDZ-containing medium. The induced shoots produced adventitious roots on MS medium supplemented with 2.5, 5, or 10 μM indole-3-butyric acid (IBA), not with indole-3-acetic acid and NAA. Although the mean number of roots per explant was not significantly different between 2.5 and 5 μM IBA, the highest rooting percentage (68%) and mean length of roots per explant (0.7 cm) was achieved at 5 μM IBA. The current study provided preliminary information toward commercial in vitro propagation of Casuarina cunninghamiana male plants.
Xiuli Shen, Vladimir Orbović, Manjul Dutt, William S. Castle, and Frederick G. Gmitter Jr.
An efficient in vitro regeneration system through direct shoot organogenesis was established for Murraya paniculata (L.) Jack (Orange Jessamine). Epicotyls, leaves, roots, and cotyledons from in vitro-germinated seedlings and several plant growth regulators (PGRs) were evaluated for their effects on plant regeneration. Longitudinally cut epicotyl segments were observed to be the optimal explants followed by uncut epicotyls (not longitudinally cut). Roots, leaves, and cotyledons were not suitable as explants as a result of little or no shoot induction. Adventitious shoot induction was enhanced by the addition of 6-benzyladenine (BA). The highest percentage of shoot induction (87%) and the greatest number of shoots per explant (12.7) occurred on Murashige and Skoog (MS) medium supplemented with 15 μM BA from longitudinally cut epicotyls followed by 5.2 shoots per explant from uncut epicotyls. Optimal concentration of gibberellic acid (GA3) for shoot elongation was observed to be 15 μM. Eighty-five percent of the regenerated shoots produced roots with an average of three roots per shoot on MS medium supplemented with 5 μM indole-3-butyric acid (IBA). Our protocol for direct shoot organogenesis can potentially lead to the development of a robust method for production of transgenic plants of M. paniculata through Agrobacterium-mediated genetic transformation.