When cotyledonary explants, excised from in vitro germinated seedlings, of pomegranate (Punica granatum L.) were incubated on solid Murashige and Skoog (1962) medium supplemented with 21 lM naptheleneacetic acid (NAA) and 9 lM 6-benzyladenine (BA), 80% of explants developed callus. A high frequency of shoot organogensis was obtained when explants were incubated on MS medium supplemented with 8 lM BA, 6 lM NAA, and 6 lM giberrellic acid (GA 3 ). However, adding 24 lM silver nitrate (AgNO 3 ) to this medium markedly enhanced shoot regeneration frequency (63%) and mean number of shoots per explant (11.26) and length of shoots (2.22 cm). Highest frequency of in vitro rooting, mean number of roots/shoot (4.32), and mean root length (2.71 cm) were obtained when regenerated shoots were transferred to halfstrength MS medium supplemented with 0.02% activated charcoal. Well-rooted plantlets were acclimatized, and then transferred to soil medium. Moreover, when zygotic embryos of P. granatum, excised from seeds collected at 16 weeks following full bloom, were incubated on MS medium containing 30 g l -1 sucrose, 15% coconut water, 21 lM NAA, and 9 lM BA, they developed the highest frequency of embryogenic callus, clumps with globular embryos, and mean number of both globular and heartshaped embryos per callus clump. Subjecting zygotic embryo explants to six-week dark incubation period was essential for embryogenic callus induction, and these were subsequently transferred to 16 h photoperiod for further growth and development of somatic embryos. Germination of somatic embryos was observed when these were transferred to MS medium was supplemented with 60 g l -1 sucrose.
Three different regeneration systems, viz. direct regeneration of adventitious shoot buds from explant, regeneration through callus cultures and somatic embryos were compared to see their effect on transfer of neomycin phosphotransferase (nptII) and beta-glucuronidase (GUS) reporter gene (gus) to Morus alba clone M5, through Agrobacterium tumefaciens mediated transformation. Pre-conditioning and co-cultivation durations had a marked effect on transformation frequency. The highest transformation frequency of 18.6% was obtained using direct induction of adventitious shoot buds. Expression and presence of transgene were assayed histochemically and through polymerase chain reaction. Southern analysis of GUS and PCR positive transformants confirmed stable integration of transgenes with two to four copy numbers. The selected transformants showed normal phenotype under in vitro and field conditions.
A protocol for plant regeneration from mesophyll and callus protoplasts of Robinia pseudoacacia L. was developed. For leaves from in vitro raised shoots, an enzyme combination of 2.0% cellulose and 0.3% macerozyme for a digestion period of 20 h resulted in the best yield of protoplasts (9.45 9 10 5 protoplast/g fresh weight). Mesophyll-derived protoplasts started cell wall regeneration within 24 h of being embedded in Nagata and Takebe (NT) medium supplemented with 5 lM NAA and 1 lM BAP followed by the first cell division on day three of culture and micro-colony (32 cells) formation within day 7-10 in the same medium. However, using callus as the starting material, a combination of 2.0% cellulose and 1.0% macerozyme for a digestion period of 24 h gave the highest protoplast yield (3.2 9 10 5 protoplast/g fresh weight). Cell wall regeneration in callus-derived protoplasts started within 24 h followed by the first cell division on the day three (96 h) and the appearance of microcolonies of more than 32 cells by the end of first week (144 h) of culture on solid WPM medium supplemented with 5 lM NAA and 1 lM BAP. Microcalli were visible to the naked eye after 45 days on solid WPM medium. Proliferation of macro-calli was successfully accomplished on solid Murashige and Skoog (MS) medium with 5 lM NAA and 5 lM BAP. Both mesophyll and callus protoplast-derived calli produced shoots on MS medium with 0.5 lM NAA and 1 lM BAP within 25-30 days and multiplied on MS medium with 1.25 lM BAP. Excised microshoots were dipped in 1-2 ml of 2.0 lM IBA for 24 h under dark aseptic conditions and transferred to double sterilized sand for rooting. The flasks containing sand were inoculated with Rhizobium for in vitro nodulation. Forty-five plants transferred to pots in the glasshouse established well.
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