Somatic embryos were induced on the roots of Camellia japonica L. plantlets regenerated from an in vitro clone of juvenile origin. The embryos appeared to differentiate from epidermic cells and to be connected with the root via a few parenchymatous cells. Somatic embryogenesis occurred on basal medium and with or without various combinations of zeatin, BA, and IBA. Secondary embryos were induced on cotyledons and/or hypocotyl regions of somatic embryos. Two morphological types of somatic embryos were developed, seed-like and bud-like types, and their formation was influenced by the presence of BA in the medium. Embryogenic capacity has been maintained for more than 24 months by subculturing secondary embryos at 7- to 8-week intervals. The best gibberellin/auxin combination for inducing the germination of isolated somatic embryos was GA at 5 mg·liter-1 G A3 and IAA at 1 mg·liter-1. P1antlets were successfully established in planting medium and have continued to grow in a greenhouse. Chemical names used: N-(phenylmethyl)-1H-purine-6-amine (BA); (1α, 2β, 4aα, 4bβ, 10β)-2,4a,7-trihydroxy-l-methyl-8-methylenegibb-3-ene-1,10-dicarboxylic acid l,4a-lactone (GA); 1 H -indole-3-acetic acid (IAA); 1 H- indole-3-butyric acid (IBA); 2-methyl-4-(1 H- purine-6-ylamino)-2-buten-l-ol (zeatin).
Ana M. Vieitez, Carmen San-José, F. Javier Vieitez, and Antonio Ballester
Guadalupe López-Puc, Adriana Canto-Flick, Felipe Barredo-Pool, Patricia Zapata-Castillo, María del C. Montalvo-Peniche, Felipe Barahona-Pérez, Nancy Santana-Buzzy, and Lourdes Iglesias-Andreu
To induce somatic embryogenesis in habanero pepper (Capsicum chinense Jacq.), the cultivar BVll-03, belonging to the red type, was used. Different explants were evaluated, as were different culture media, the composition of which varied in the content of plant growth regulators. Results showed the formation of somatic embryos from cotyledons, zygotic embryos, germinated zygotic embryos, hypocotyls, and cotyledonary leaves. Explants were cultured on Murashige and Skoog medium supplemented with 2,4-D (9.05 μm). The somatic embryos always formed directly from the explant, without callus formation, and the greatest efficiency was obtained when segments of hypocotyls were cultured, obtaining 175 ± 20 somatic embryos per explant. Only the somatic embryos obtained on Murashige and Skoog medium containing 2,4-D (9.05 μm) and treated with abscisic acid (ABA) (1.89 μm) before their transfer to the germination media (Murashige and Skoog + 1.1 μm GA3) emitted their radicule and expanded their cotyledonary leaves (60%), whereas the remaining embryos did not achieve germination because of different causes (abnormalities, delayed development). Not only is this protocol of somatic embryogenesis the first to be reported for this species (C. chinense Jacq.), but it is also the most efficient reported so far, within the Capsicum genus.
Jane Kahia, Margaret Kirika, Hudson Lubabali, and Sinclair Mantell
production of Ruiru11 planting materials by using alternative methods such as tissue culture. A great deal of work has been carried out on coffee somatic embryogenesis using the indirect method. However, direct somatic embryogenesis (DSE) has not received
A. Levi and K.C. Sink
The histology and morphology of developing asparagus Asparagus officinalis L.) somatic embryos arising in callus cultures were examined and contrasted with that documented for zygotic embryos. Histological sections of lateral bud-derived callus cultured for 2 weeks on embryo induction medium consisting of Murashige and Skoog salts and vitamins (MS) with 1.5 mg NAA/liter and 0.1 mg kinetin/liter indicated the formation of distinct groups of embryogenic cells. At 4 weeks, the callus was comprised of embryos in the early and late globular stages and a few bipolar embryos. Within 2 weeks on embryo development medium consisting of MS with 0.05 mg NAA/liter and 0.1 mg kinetin/liter, the globular embryos developed a bipolar shape having an expanded upper region that formed the cotyledon and a smaller region that formed the radicle. Within 4 to 6 weeks on this latter medium, each mature bipolar embryo was opaque and had a large cotyledon, a distinct shoot apex at the cotyledon-hypocotyl junction, and vascular connections between the radicle, shoot apex, and cotyledon. Many mature somatic embryos resembled the asparagus zygotic embryos in having a crescent shape, whereas others had a short but wide cotyledon. Both somatic embryo types converted to plantlets at equal rates. Chemical names used: N- (2-furanylmethyl)-1 H -purin-6-amine (kinetin); 1-naphthaleneacetic acid (NAA).
I. Oiyama and S. Kobayashi
Some undeveloped seeds from mature Citrus fruit of monoembryonic diploid cultivars crossed with a tetraploid selection were observed to be polyembryonic. The multiple embryos formed a small mass the the micropylar end. Plants regenerated in vitro from the embryos in polyembryonic seeds were triploid and showed identical peroxidase banding patterns on acrylamide gels. These results indicate that the multiple embryos found in the undeveloped seed from monoembryonic diploid × tetraploid crosses are genetically identical and of zygotic origin.
Judith Corte-Olivares, Gregory C. Phillips, and S.A. Butler-Nance
V.M. Gingas and B.D. Stokes
S.A. Merkle and B.A. Watson-Pauley
Bigleaf magnolia (Magnolia macrophylla Michx.) cultures were initiated from immature seeds on an induction medium containing 9.0 μm 2,4-D, 1.1μm BA, and 1 g casein hydrolysate/liter. After 2 months on induction medium, one culture produced adventive embryos. Clumps of embryos transferred to liquid induction medium proliferated as nodules, which grew in diameter, but failed to produce embryos while maintained in induction medium. Nodules transferred to basal medium produced clumps of somatic embryos, which continued to produce repetitive embryos with monthly transfer to fresh basal medium. Individual embryos transferred to basal medium lacking casein hydrolysate germinated and leaves expanded. Plantlets derived from these embryos were transferred to potting mix and acclimatized to greenhouse conditions. Chemical names used: (2,4-dichlorophenoxy)acetic acid (2,4-D); N -(phenylmethyl)-lH-purin-6-amine (BA).
Christopher S. Cramer and Mark P. Bridgen
Disinfected midrib sections of Mussaenda `Queen Sirikit' ≈3 to 4 mm in size were cultured on a basal medium of Murashige and Skoog salts and vitamins, 87.7 mm sucrose, and 5 g Sigma agar/liter supplemented with several concentrations of indole-3-acetic acid (IAA) (0, 5.0, 10.0, 20.0 μm) and 6-benzylaminopurine (BAP) (0, 0.5, 1.0, 2.5, 5.0, 10.0, 25.0, 50.0 μm). Cultures were subculture onto the same treatment after 5 weeks and observed weekly for 15 weeks for the presence of somatic embryos. As somatic embryos were produced, they were subculture onto basal medium supplemented with 0.5, 1.0, 2.5, or 25.0 μm BAP. Callus was first observed at 2 weeks in cultures grown on basal medium supplemented with 5.0–20.0 μm IAA and 0–50.0 μm BAP. Somatic embryos were observed at 8 weeks on basal medium supplemented with 5.0–10.0 μm IAA and 2.5–5.0 μm BAP. Callus cultured on 0–10 μm IAA and 5.0–10.0 μm BAP produced the greatest number of somatic embryos by 15 weeks. Somatic embryos subculture to basal medium supplemented with 25.0 μm BAP proliferated shoots, while eliminating BAP from the medium resulted in root and callus production. Shoots and entire plants were removed from in vitro conditions and successful] y acclimated to greenhouse conditions. Somatic embryo-derived plants flowered sporadically 25 to 35 weeks after removal from in vitro conditions. Variations in sepal number and leaf number per node were observed at 1% to 5%.
S.A. Merkle and A.T. Wiecko
Cultures were initiated from immature seeds of three species of magnolia: sweetbay magnolia (Magnolia virginiana L.), fraser magnolia (M. fraseri Walt.) and yellow cucumbertree [M. acuminata var. cordata (Michx.) Sarg.]. Immature seeds were bisected longitudinally and cultured on a solidified conditioning medium containing 2 mg 2,4-D/liter, 0.25 mg BA/liter, 40 g sucrose/liter, and 1 g casein hydrolysate/liter. Cultures were maintained in the dark at 22C and transferred to fresh medium at monthly intervals. Within 2 months of culture, somatic embryos or proembryogenic masses proliferated from one end of the endosperm mass. Somatic embryos and proembryogenic masses of each species were cultured on a hormone-free version of the conditioning medium to complete maturation and then transferred to the same hormone-free medium, minus casein hydrolysate, to initiate germination. Germinants were transferred to a hormone-free plantlet development medium for conversion. Plantlets of all three species survived transfer to soil mix and continued to grow. Chemical names used: (2,4 -dichlorophenoxy) acetic acid (2,4-D), N- (phenylmethyl)-1H-purin-6-amine (BA).