Cotyledon explants were harvested from immature walnut fruits during July and August 1991. Media consisted of either WPM with 0.1 μM 2,4-D, 5.0 μM TDZ and 1.0 g/liter casein hydrolysate or DKW with 4.4 μM BA, 0.05 μM IBA, 9.3 μM Kinetin and 250 mg/liter l-glutamine. Treatments were arranged factorially with 2 gelling agents, 7 g/liter Sigma agar or 2 g/liter Gelrite and were incubated in light or in darkness. After 4 weeks, all explants were placed on basal DKW with no growth regulators and were cultured in darkness. The best treatment tested was from seeds collected 14 weeks post-anthesis on WPM, agar, and incubation in light (22 embryos/explant, 78% embryogenesis). Use of DKW and gelrite in darkness resulted in 1 embryo/explant and 38% embryogenesis. Up to 90% shoot organogenesis also occurred on cotyledon explants from seeds collected 16 weeks post-anthesis and placed on WPM. Shoots elongated on stationary liquid DKW with 10 μM BA.
Lynn M. Long, John E. Preece, Gerald R. Gaffney, and J. W. Van Sambeek
Immature leaf laminae and petioles of `Regale' and `Fry' muscadine grapes (Vitis rotundifolia Michx.) were cultured on Nitsch and Nitsch (NN) medium supplemented with 9.0 μm 2,4-D and 4.4 μm BA, and gelled with agar. Callus and original explant tissues were transferred to NN medium containing 10.7 μm NAA and 0.9 μm BA to proliferate embryogenic callus, which, when transferred to NN medium without growth regulators, yielded globular embryos. The embryos matured and germinated after being subcultured to fresh medium without growth regulators. Somatic embryogenesis incidence was greater from petioles than laminae: 90% of `Regale' and 50% of `Fry' petioles formed embryos, compared with 14% and 2% of laminae, respectively. Culturing germinated somatic embryos on NN medium with 1 μm BA enhanced shoot growth. Regenerated plants flowered and appeared morphologically normal. Chemical names used: N-(phenylmethyl)- 1H -purin-6-amine (BA); 2,4-dichlorophenoxyacetic acid (2,4-D); α- naphthaleneacetic acid (NAA).
S.N. Talhouk, M. Shmoury, R. Baalbaki, and S. Khuri
Somatic embryogenesis offers a great potential for large-scale production of Cedrus libani, which is important not only as a forest tree, but also for the development of a timber industry. In an attempt to optimize conditions for embryogenic callus induction, we used zygotic embryos at different developmental stages as explants, compared different media, and used several hormone levels and combinations. Results indicated that post-cotyledonary immature embryos had highest induction efficiency. Four different media namely 1/2 MS, Durzan, Litvay's, and Von Arnold supplemented with similar hormone levels showed no significant difference in efficiency of callus induction. Induction frequencies of embryogenic callus from explants subjected to different hormone levels and combinations were dependent on the developmental stage of the explant.
Y. Mohamed-Yasseen, T. L. Davenport, W. E. Splittstoesser, and R. M. Skirvin
A method for regeneration of somatic embryogenesis from witloof chicory is described. Explants were taken from leaf veins of stored witloof chicory. Internal bacterial infection was found in 100% of the leaf bases but decreased gradually toward the leaf tips. Bacterial free explants were taken from the distal third and cultured on Murashige and Skoog medium (MS) containing 1.3 uM 2,4-D, 1.3 uM kinetin, and 100 mg/L casein hydrolysate. A pale yellowish, nodular callus formed after 4 weeks and were maintained in the same medium for 8-12 months with one change to a fresh medium every 4 weeks. Callus were suspended in the same medium without agar for 4-6 weeks with one change to a fresh medium every 2 weeks. Embryo-like structure appeared upon transfer to MS liquid medium containing 1.8 uM benzyladenine. Embryo germination was accomplished in 1/4 strength of MS medium with 01 without 1 g/L activated charcoal.
Partially expanded male catkins of swamp white oak (Quercus bicolor Willd.) and red oak (Quercus rubra L.) were cultured on Murashige and Skoog (MS) medium supplemented with BA or 2,4-D. Explants on 2,4-D produced a yellow embryogenic callus originating from the junction of the pedicel and peduncle. Subsequent transfers to MS with BA and then MS without growth regulators resulted in callus proliferation. After 10 to 14 weeks in culture, white embryoids developed from the callus of Q. bicolor. Separated and individually cultured embryoids underwent direct, repetitive embryogenesis. Upon transfer to l/2-strength MS, embryoid germination and plant regeneration occurred. Callus of Q. rubra degenerated after 5 months in culture, failing to yield embryogenic structures. Chemical names used: dichlorophenoxyacetic acid (2,4-D); benzyladenine (BA).
Bipul Biswas, Nirmal Joshee, Ashish Yadav, and Anand K. Yadav
Phalsa[Grewia asiatica (L.) Tiliaceae] is an exotic fruit with good nutraceutical values. It cannot be grown in temperate climates with severe winters. Therefore, genetic improvement of phalsa for cold tolerance is essential. In order to apply biotechnology through genetic transformation to enhance cold hardiness, a reliable and rapid micropropagation system is needed. Thus, developing the most dependable micropropagation protocols for phalsa was the primary goal of this research. Phalsa explants prepared from different tissues, including leaf, nodes, internode, and zygotic embryos, were collected from mature trees growing in the specialty plants house, cultured on MS medium supplemented with various cytokinins alone or along with auxins and incubated under a 10-hour photoperiod at ambient temperature. In vitro propagation of phalsa tissues through both organogenesis and somatic embryogenesis was achieved. Of these, single shoots were developed from nodal explants as a result of budbreak on MS medium supplemented with BAP, kinetin, and zeatin separately. Somatic embryos were developed from the zygotic embryos when cultured on MS medium with 0.023 μm BA + 0.022 μm zeatin, for 2 weeks following a pulse treatment on NN medium supplemented with 5% sucrose, 0.11 μm BAP, 0.22 μm 2,4-D, and 29.20 μm L-glutamine. Somatic embryogenesis was also observed on modified basal medium supplemented with 13% sucrose, 58.40 μm L-glutamine, and 1.75 μm IAA. Enormous callusing was a major problem for in vitro studies with this species, irrespective of media composition. Further studies for multiple shoot development and higher frequency of SE induction are under way.
Mary W. George and Robert R. Tripepi
Previous reports of somatic embryogenesis on rose tissues involved an embryogenic callus stage with either a complicated multi-step process or low numbers of embryos being produced. We have produced somatic embryos without a callus stage from leaf explants of the cut rose cultivar `Golden Emblem' by using a two step process. Explants were obtained from microshoots of `Golden Emblem' that had been in culture for three years. All experiments were repeated twice. When explants were maintained on Murashige and Skoog (MS) with 0.4 μM NAA and 0.4 μM kinetin for 10 weeks, 10% or less of the explants produced somatic embryos. Keeping the explants on the NAA/kinetin medium for two weeks, then switching to medium with 0, 0.5, 1.0, or 10.0 μM kinetin for the remaining 8 weeks failed to increase embryo production. Decreasing the time the explants were on the NAA/kinetin medium to 8 or 12 days, and then placing explants on MS medium with 1.0 μM kinetin increased somatic embryo production to a maximum of 25%. By limiting the length of time the rose leaf explants were exposed to auxin, direct somatic embryo production was increased.
Yuexin Wang, Zoran Jeknić, Richard C. Ernst, and Tony H.H. Chen
A protocol was developed for efficient plant regeneration of Iris germanica L. `Skating Party' from suspension cultures. Suspension cultures were maintained in Murashige and Skoog (MS) basal medium (pH 5.9) supplemented with 290 mg·L–1 proline, 50 g·L–1 sucrose, 5.0 μm 2,4-D, and 0.5 μm Kin. Suspension-cultured cells were transferred to a shoot induction medium (MS basal medium supplemented with 10 mg·L–1 pantothenic acid, 4.5 mg·L–1 nicotinic acid, 1.9 mg·L–1 thiamine, 250 mg·L–1 casein hydrolysate, 250 mg·L–1 proline, 50 g·L–1 sucrose, 2.0 g·L–1 Phytagel, 0.5 μm NAA, and 12.5 μm Kin). Cell clusters that proliferated on this medium differentiated and developed shoots and plantlets in about 5 weeks. Regeneration apparently occurred via both somatic embryogenesis and shoot organogenesis. A series of experiments was conducted to optimize conditions during suspension culture to maximize subsequent plant regeneration. Parameters included 2,4-D and Kin concentrations, the subculture interval, and the size of cell clusters. The highest regeneration rate was achieved with cell clusters ≤280 μm in diameter, derived from suspension cultures grown for 6 weeks without subculturing in liquid medium containing 5 μm 2,4-D and 0.5 μm Kin. Up to 4000 plantlets with normal vegetative growth and morphology could be generated from 1 g of suspension-cultured cells in about 3–4 months. Chemical names used: 2,4-dichlorophenoxyacetic acid (2,4-D); kinetin (Kin); 1-naphthaleneacetic acid (NAA).
Nian-Oing Shi and Zong-Ming Cheng
Some antibiotics mimic plant hormones on cell growth and plant regeneration. Cefotaxime and carbenicillin were tested in American elm for induction of embryogenesis from cotyledonary explants, which normally show organogenesis. Cotyledons from 1-week-old in vitro germinated seedlings were placed on a shoot regeneration medium (a modified MS medium containing 15 μ M BA, B5 vitamins and 0.3% gelrite) with various levels of cefotaxime and carbenicillin. One hundred percent of explants showed embryogenesis in the medium supplemented with 125 μg/ml cefotaxime; 75% explants regenerated somatic embryos in medium with 500 μg/mg carbenicillin; and only 50% explants produced somatic embryos in the medium with both of these antibiotics. In control medium without antibiotics, 100% explants regenerated shoots, instead of somatic embryos. Further studies are necessary to determine the nature of these antibiotics on shifting developmental pathways and their stimulatory effect on embryogenesis from American elm cotyledons.
Isabel Arrillaga, Victoria Lerma, Pedro Pérez-Bermúdez, and Juan Segura
The effects of stage of microspore development, cold pretreatment, and growth regulators on the embryogenic response of anthers from service tree (Sorbus domestica L.) are reported. Callus proliferation required the presence of growth regulators in the culture medium, best results being obtained with combinations of auxins and BA. Microspore embryogenesis was only induced when anthers containing tetrads or uninucleate pollen were cultured on BA-supplemented media containing IBA or IAA. A cold pretreatment before anther culture elicited a significant decrease in callus formation and inhibited embryogenesis. Chemical names used: benzyladenine (BA), indole-3-butyric acid (IBA), indole-3-acetic acid (IAA).