Douglas fir megagametophyte development in situ and in vitro




Chiwocha, Sheila

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Megagametophyte development in situ and in vitro was investigated in Douglas fir to address the following questions: (1) Do endogenous levels of plant hormones change during megagametophyte development and are they associated with morphological changes? (2) Can megagametophytes be cultured prior to fertilization? (3) Can embryos be rescued from megagametophytes cultured soon after fertilization? A histochemical study of storage reserve deposition during megagametophyte development was performed with material isolated weekly for 11 weeks. Prior to fertilization, starch was detected in the neck cells of megagametophytes analyzed 9 weeks after pollination (WAP). During embryogenesis, starch was deposited in the central region of megagametophytes. Proteins and lipids were first detected in the prothallial cells in the periphery of megagametophytes isolated 14 and 15 WAP, respectively. With further development, starch was deposited in prothallial cells around the corrosion cavity, while proteins and lipids were spatially localized to prothallial cells in the periphery. In the embryo, starch accumulation was preferentially localized in the root cap and the embryonal suspensor cells at 17 WAP. A parallel study quantifying the endogenous levels of plant hormones: IAA, IAAsp, Z, ZR, iP, IPA, ABA and ABA-GE, in megagametophytes was performed. Hormones were extracted, purified and fractionated using HPLC. To correct for losses due to procedures, radiolabelled standards were added prior to extraction. The hormones were quantified using an ELISA method. On a dry weight basis, Z levels were highest in megagametophytes at the late central cell stage (8 WAP). During embryogenesis, Z levels peaked during week 13. ZR peaked twice at 13 and 17 WAP. The iP content of megagametophytes increased at 10, 13 and 17 WAP while iP.A concentration increased at 13 and 17 WAP. Prior to fertilization, the free LAA was highest in megagametophytes at 9 WAP. During embryogenesis, the major lA A accumulations occurred at I I ,13 and 15 WAP. lAAsp concentrations reached their highest levels at 10, 14 and 18 WAP. ABA content increased at 11, 13 and 17 WAP. In contrast, ABA-GE levels were relatively constant over the 11 weeks analyzed. Megagametophytes were isolated weekly from 7–10 WAP and cultured on a modified half-strength Litvay's medium supplemented with one of three auxins (NAA, IBA or 2,4-D) and a cytokinin (2 mg/L BAP). Each auxin was tested at three levels: 0.1, 1.0 or 10 mg/L. The objective was to determine whether the megagametophytes would continue to grow in culture. Megagametophytes increased in length after 9 and 18 days of culture. Auxin and cytokinin supplements had a significant effect on growth for material isolated 7 or 10 WAP. However, the viability of the archegonia rapidly declined on all the media tested. The most optimal treatment for each auxin type (BAP in combination with 0.1 mg/L NAA. 1.0 mg/L IBA or 1.0 mg/L 2.4-D) was used to initially culture pollinated megagametophytes in the embryo rescue experiment. After 21 days, megagametophytes were transferred to media containing ABA concentrations of 0.5, 20 or 40 μM. A majority of the rescued embryos were developmentally arrested at the globular stage. Only three embryos, containing over 30 cotyledons each, matured on ABA concentrations of 5, 20 or 40 μM. In conclusion, the prothallial cells of the pre-fertilization megagametophytes could be cultured for long periods and their growth was not dependent on the presence of viable archegonia. The endogenous levels of plant hormones varied with megagametophyte development and were associated with morphological changes. This information has implications for growing megagametophytes for in vitro fertilization and embryo rescue experiments. The endogenous levels of plant hormones could be used to design culture media for rescuing embryos resulting from in vitro fertilization in Douglas fir.



Douglas fir, Plant embryology, Forest genetics