An improved and simplified somatic embryogenesis protocol in Chir pine (Pinus roxburghii)

Forests are considered to be an important pillar for a country's bioeconomy but unfortunately are being depleted globally. Pinus roxburghii has considerable ecological and economic importance for Pakistan. It is successfully used as a pioneer species in afforestation and reforestation programs in the era of climate change. This paper reports on a significantly improved protocol for P. roxburghii somatic embryogenesis. Successive developmental stages for zygotic as well as somatic embryos were reported first time for this important pine species. Initiation of somatic embryogenesis was affected by the developmental stage of the zygotic embryo. Immature pre-cotyledonary zygotic embryos produced 33% initiation of embryonic masses on LP-889 medium supplemented with 11 µM Naphthaleneacetic acid (NAA), 2.5 µM Benzylaminopurine (BAP), 2.5 µM kinetin and 4 µM Abscisic acid (ABA). LP-1250 medium provided with 2 µM BAP, 2 µM kinetin, 5 µM 2, 4-Dichlorophenoxyacetic acid (2, 4-D) and 5 µM ABA was used for the proliferation and maintenance of embryogenic tissue. Polarized immature somatic embryos were clearly visible during the proliferation and maintenance phase. Two media; mLV and LP-1562 were tested as well as several combinations of L-glutamine and ABA for maturation. Maximum number of somatic embryos (310 embryos per g embryogenic tissue) was obtained on LP-1562 medium (containing 450 mg/l of L-glutamine and 20 µM ABA) as compared to 204 embryos per g embryogenic tissue on mLV medium (containing 900 mg/l L-glutamine and 60 µM ABA). Mature somatic embryos were formed on maturation medium after 6-8 weeks. Successful conversion of somatic embryos resulted after 12 weeks on LP-397 medium. However, low rooting rates (7% and 3%) were observed for somatic embryos formed on maturation media, i.e. LP-1562 and mLV, respectively and precocious germination of the mature somatic embryos still remains a serious constraint.