Somatic embryogenesis and organogenesis in callus cultures of a tree legume — Albizia richardiana King

Hypocotyl explants of 1 and 10 mm lengths were excised from 12-day-old in vitro-grown seedlings of Albizia richardiana. The larger pieces, after 40 days of culture, developed shoots along with green calli on B₅ + BAP (10^–7–10^–5M), while the smaller segments produced only green calli on B₅+BAP (10^–7–10^–4M) medium. Some of the green calli turned morphogenic and started producing somatic embryos with the 2nd sub-culture and shoots from 7th sub-culture onwards. Calli retained the morphogenic potential even after repeated sub-culturing for over two years. The number of embryos in an embryogenic culture varied from 2 to 20 per callus mass of 5–6.5 cm³. Sucrose at the 2% level in MS medium was optimal for embryogenesis while 4% was optimal for shoot bud differentiation. Higher levels of sucrose (6–10%) caused browning of green calli and also inhibited differentiation into embryos and shoot buds. By selective sub-culturing of 0.1 cm³ pieces of embryogenic calli on MS+10^–5M BAP, 46% of the cultures produced somatic embryos. The latter germinated into plantlets on Knop's medium.Abbreviations BAP 6-benzylaminopurine - B₅ Gamborg et al., 1968 medium - IAA Indole-3-acetic acid - MS Murashige and Skoog's (1962) medium     

In vitro culture of embryos of Cucumis spp.: heart-stage embryos have a higher ability of direct plant formation than advanced-stage embryos

Summary The ability of embryos at different developmental stages to form plants in vitro has been studied in cultivated Cucumis sativus L. and in the wild species C. zeyheri 2 x Sond. and C. metuliferus Naud. On MS medium containing 3.5% sucrose, 0.1 mg 1^–1 kinetin (Kn) and 0.01 mg 1^–1 indoleacetic acid (IAA), proembryos (0.03–0.05 mm) and early globular embryos (0.05–0.08 mm) from the wild species developed into plants in low frequencies of 8% and 21%, respectively. These embryos should be surrounded by the embryo sac tissue. On the same medium late globular (0.08–0.1 mm) and early heart-stage embryos (0.1–0.3 mm) developed into plants in moderately high and high frequencies of 48% and 83%, respectively. The presence of the embryo sac at these stages was still beneficial, but no longer a prerequisite. Late heart-stage embryos (0.3–0.8 mm) also showed high frequencies of plant formation, 63%, if Kn was applied at a concentration of 1 mg 1^–1. From the early cotyledon stage onwards, the frequency of plant formation gradually decreased, reaching a minimum at the late cotyledon stage. Subsequently it began to increase again up to the late maturation stage. The poor plant formation shown by the intermediate-aged embryos could be improved slightly by lowering the sucrose concentration to 0.5% and by increasing the Kn concentration to 10 mg 1^–1. Relative to the wild species, embryos of C. sativus showed lower percentages of plant formation. The optimum sucrose concentration was 2% for the heart-stage C. sativus embryos. In all three species the ability to form plants strongly decreased with increasing embryo age, from early to late cotyledon. This is thought to be caused by the increasing tendency of the embryos at these stages to continue in vitro the normal embryo development.     

The development of onion (<Emphasis Type="Italic">Allium cepa</Emphasis> L.) Embryo sacs <Emphasis Type="Italic">in vitro</Emphasis> and gynogenesis induction in relation to flower size

Summary The development of embryo sacs (ES) in vitro and induction of gynogenesis were studied in onion flower bud culture. Explants were divided into three groups according to their size at inoculation: (a) small flower buds (2.3–3.0 mm in diameter); (b) medium flower buds (3.1–3.7 mm); and (c) large flower buds (3.8–4.4 mm). For histological study, excised ovaries were fixed at inoculation and then at 3-d intervals until day 12, and after 2 and 3 wk of culture. Some explants were cultured until embryo emergence, i.e., 3–5 mo. In total, 2592 ovules were examined histologically. At inoculation, 83% of ovules in small flower buds contained a megaspore mother cell; in 17% of ovules, two-nucleate ES occurred. In medium flower buds two-nucleate, four-nucleate, and mature ES were present at frequencies of 15%, 46%, and 40%, respectively. In large flower buds, only mature ES occurred. In vitro conditions did not disturb meiosis and megagametophyte development in non-degenerated ovules. Regardless of the developmental stage at inoculation, only mature ES occurred on day 12. Gynogenic embryos were found after 2 wk of culture, indicating that embryos developed in mature ES exclusively. Embryos were detected in 5.4% of histological studied ovules; however, the number of embryos after 3–5 mo. was higher (12.4%). The parthenogenetic origin of the embryos is discussed. In addition, ES containing endosperm only (6.5%) and both endosperm and embryo (0.4%) were observed.     

In vitro culture of isolated microspores and regeneration of plants in Brassica campestris

A protocol previously developed for B. napus microspore culture was modified to produce embryos from several lines of Brassica campestris. Bud size, genotype, media constituents, and incubation time and temperature were examined. Donor plants were grown in a growth cabinet at a day/night temperature of 10/5°C. Microspores were isolated from buds 2.0 – 2.9 mm in length and cultured in modified Lichter (1982) medium containing 17% sucrose, pH 6.2. After 48 h at 32°C, the incubation medium was replaced with NLN (Lichter 1982) medium containing 10% sucrose. Microspores were cultured at 24°C in darkness and embryos developed after three weeks. More than 1000 plants have thus far been regenerated. Genotypic differences were observed for microspore embryogenesis. The majority of the regenerants were haploid, however colchicine could be effectively used to achieve chromosome doubling.     

On the Embryonic Development and Ovule Culture of Interspecific Hybrids Between Populus simonii Carr and P.pyramidalis Borkh

The embryonic development following P. simonii Cart. × P. pyramidalis Borkh. is described in the present paper. The majority of pollen grains of P. pyramidalis Borkh. may germinate on the stigma of P. simonii Cam and the pollen tubes grow normally through the style and enter the embryo sac from the micropyle. Fertilization occurs as usual 4–7 days after pollination. A lot of proembryos and heart-shaped embryos are abortive; however, the others may develop normally and grow into mature embryos. Some of the endosperms appear normal and others may degenerate at free nuclear stage or cease to develop further at cellular stage. The ovules containg immature hybrid embryos of 19 days, 22 days, 26 days and 29 days after pollination at various developmental stages (heart-shaped stage, torpedo-stage and cotyledonary elongation stage) are excised and inoculated on nutrient agar for culture. The results show that: ( 1 ) Mll0 medium ( 1/2 MS+IAA 0.01 mg/L+BA 0.1 mg/L+sucrose 2% ) is the best of all the media used; (2) immature hybrid embryos of various developmental stages contained in ovules cultured in vitro may grow into normal plantlets.     

Somatic embryogenesis and plant regeneration from protoplasts of asparagus (Asparagus officinalis L.)

Protoplasts were isolated from embryogenic calli of Asparagus officinalis L. cv. Mary Washington and cultured in 1/2 MS medium with 1 mg/l NAA, 0.5 mg/l zeatin, 1 g/l L-glutamine, 0.6 M glucose and 0.1% Gellan Gum. Protoplasts started to divide after 3–4 d of culture and formed visible colonies after 30 d of culture. The percentage of colony formation (plating efficiency) was 7.2%. The colonies were then transferred onto Gellan Gum-solidified MS medium containing 1 mg/l 2,4-D and 3% sucrose for further growth. Somatic embryos were induced from all colonies of 0.5–1.0 mm size after transferring to 1/2 MS medium lacking growth regulators. After treating these somatic embryos (1–3 mm) in distilled water for a week, 30–40% of them germinated normally and grew into plantlets 20–30 d after transplanting on 1/2 MS medium containing 1 mg/l IBA, 1 mg/l GA₃ and 1% sucrose. These protoplast-derived plants were diploid with 20 chromosomes.Abbreviations BA 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - GA₃ gibberellic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (1962)     

A new approach to direct somatic embryogenesis in Medicago

A highly efficient system for direct somatic embryogenesis is described. Leaf sections originating from young trifoliate leaves of Medicago falcata line 47/1–5 and Medicago sativa line No2/9R, directly produced embryos after cultivation in liquid B5IV induction medium. In comparison with indirect somatic embryogenesis the system omits the callus stage and thus allows shortening of the process of somatic embryogenesis in alfalfa by 35–40 days. It permits the avoidance of secondary changes occurring during the process of dedifferentiation. A modified B5/3H medium containing Polyethylene Glycol 6000 promoted embryo development from globular up to torpedo stage. It was clearly shown that 2.5% Polyethylene Glycol stimulated this process for both H. falcata 47/1–5 and M. sativa No 2/9R. Maturation of torpedo stage embryos was carried out on solidified or liquid abscisic acidcontaining medium. A 30M abscisic acid concentration was optimal in allowing one embryo to yield one plant. Somatic embryo conversion to plants and plant regeneration was performed on Murashige and Skoog medium. Regenerated plants showed a normal morphology.Abbreviations ABA Abscisic acid - B5 Medium of Gamborg et al.(1968) - COT Cotyledone stage embryos - 2,4-D 2,4-dichlorphenoxyacetic acid - FW Fresh weight - GA3 Gibberellin A3 - MS Medium of Murashige and Skoog (1962) - PEG Polyethylene Glycol - POLY Polyembryos     

Isolation,culture and division of olive (Olea europaea L.) protoplasts

Protoplasts from Olea europaea L. have been compared in terms of their yield, viability, cell division and callus differentiation. Viable protoplasts were isolated from in vitro cultured leaves and cotyledons by an overnight incubation in an enzyme solution containing 1–1.5% driselase and 0.5M sucrose. This method allowed high yield of purified protoplasts, which floated and formed a dark green band at the meniscus, after centrifugation. Purified protoplasts were diluted to 3×10⁴ protoplasts·ml^–1 in culture medium. After cell wall regeneration, protoplasts gradually increased their volumes under appropriate conditions. The first divisions occurred during the second week in culture. Division efficiency ranged from 5.2 to 9.8% after 20 days in culture. Two weeks later visible microcolonies developed only from cotyledon protoplasts. After 6 weeks in culture, the microcalli were transferred to a solidified culture medium with 0.6% agarose, which induced active callus growth.Abbreviations OM olive proliferation medium, Rugini 1984 - Omg OM for the germination of olive embryos - OMr=OM for root induction - OMp=OM for protoplasts - OMc=OM for callus - BN Bourgin and Nitsch medium 1967 - IBA indol-3-butyric acid - NAA naphthalene acetic acid - 2,4-D dichlorophenoxyacetic acid.     

Plant regeneration from immature embryos of peanut

Plant regeneration from immature embryos of peanut (Arachis hypogaea L.) can be accomplished through somatic embryogenesis. The highest frequency of somatic embryo formation occurred on B5 medium plus 0.5–1.0 mg/l picloram. Shoots and plants developed from the somatic embryos only after extended culture on basal medium. Shoots were excised from thick embryonic roots and rerooted on Murashige and Skoog medium containing half the normal concentration of inorganic salts. This technique should be useful for the production of interspecific hybrid plants from immatureArachis embryos.     

Induction of freezing tolerance in microspore-derived embryos of winter Brassica napus

Freezing tolerance was induced in microspore derived embryos of winter Brassica napus cv. Jet neuf by the addition of ABA or mefluidide to the culture media during embryogenesis. Survival after freezing was estimated by culture of frozen-thawed embryos to plantlets. A higher freezing tolerance (50% survival at –15°C) was induced when 50 M ABA or 3.2 M mefluidide was incorporated initially into the medium during embryogenesis at 25°C followed by culture at 2°C for 3 weeks. When embryos were induced in the absence of ABA or mefluidide and maintained at 2°C for even as long as 12 weeks a lower degree of freezing tolerance (10% survival at –15°C) was obtained. Plants regenerated from embryos hardened maximally by a combination of either ABA or MFD with low temperature did not require further vernalization for flowering.Abbreviations ABA abscisic acid - MFD mefluidide - 2,4-D 2,4-dichlorophenoxyacetic acid - LT₅₀ killing temperature for 50% of the embryos     

Somatic embryogenesis in Encephalartos cycadifolius

Callus cultures of Encephalartos cycadifolius were established from zygotic embryo explants on a modified B5 medium containing 1 mg l^–1 2,4-D and 1 mg l^–1 kinetin. Callus was transferred to media containing various combinations of 2,4-D and kinetin for improvement of somatic embryogenesis. Somatic embryos were produced on media with several growth regulator combinations. The somatic embryos developed from proembryos, which developed long suspensors. A dicotyledonary embryo formed at the distal end of the suspensor. The embryos turned green in light. When transferred to a medium containing 1 mg l^–1 ABA the somatic embryos matured. The suspensors desiccated and these embryos rooted when transferred to a medium without phytohormones.Abbreviations ABA abscisic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Induction of autonomous endosperm in Lupinus luteus,Helleborus niger and Melandrium album by in vitro culture of unpollinated ovaries

Autonomous division of the endosperm was induced by in vitro culture of unpollinated ovaries or placenta-attached ovules in Helleborus niger, Lupinus luteus and Melandrium album. The induction frequencies for the three species were 50%, 10–20% and 0.1%, respectively. The endosperms contained up to 20 free nuclei; only a few ovules with 80–420 endosperm nuclei were found. Induction of autonomous division of the endosperm, which is unusual in amphimictic plants, was observed in three new species. No embryos appeared in the ovules. This suggests a developmental independence of the endosperm from the embryo in the culture of unpollinated ovaries or ovules.     

In-vitro culture of fertilized embryo sacs of maize: Zygotes and two-celled proembryos can develop into plants

Fertilized embryo sacs of Zea mays L. surrounded by a few layers of nucellar cells were cultured in vitro. Primary expiants contained zygotes or twocelled proembryos. Embryos of various sizes and shapes were isolated from 12–48% of explants after two weeks of culture in hormone-free media supplemented with 6–12% of sucrose. Many embryos were at the transition or proembryo stages whilst the rest were either differentiated, with a scutellum, a coleoptile and a shoot apex, or had a deformed apical part. Organogenesis started in 36–89% of embryos cultured on a semisolid medium supplemented with coconut water. Most of the embryos formed only roots but up to 9% of embryos regenerated into plants. This simple method leads the way to plant regeneration from in-vitro-manipulated zygotes or proembryos of maize.Abbreviation NBM medium composed of N6 macronutrients, B5 micronutrients and MS vitamins This research was supported by an I.N.R.A. post-doctoral fellowship. The authors thank R. Blanc for donor plant culture, Dr. M. Cock (Reconnaissance Cellulaire et Amélioration des Plantes, Université Lyon 1) for correction of the English and P. Audenis for micrograph development.     

In-ovulo embryo culture and seedling development of cotton (Gossypium hirsutum L.)

A convenient and reliable method for culturing cotton embryos is needed to obtain interspecific hybrids of this genus. C.A. Beasley and I.P. Ting (Amer. J. Bot. 60, 130, 1973) developed a phytohormone-supplemented medium (BTP) upon which the growth of ovules was similar that of in situ ovules. This medium was examined for in-ovulo embryo culture. Although good ovule growth occurred on BTP no embryos developed to maturity. However, when the medium was supplemented with NH ₄ ⁺ , more than 50% of the ovules produced mature embryos, and many of these germinated precociously after 8–10 weeks of culture. After germination seedlings were established on a separate medium designed to give balanced root and shoot growth. Subsequently young plants could be transferred to pots for greenhouse culture.     

Isolated microspore culture of Chinese flowering cabbage (Brassica campestris ssp. parachinensis)

Microspores of several genotypes of Brassica campestris ssp. parachinensis have been cultured in vitro and induced to undergo embryogenesis and plant formation. Conditions favourable for embryogenesis in this species include a bud size of 2–2.9 mm, NLN-13 culture medium (Nitsch and Nitsch 1967; Lichter 1981, 1982; Swanson 1990), and an induction through exposure to 32°C for a period of 48 h. Longer periods of an elevated temperature for induction of embryogenesis resulted in embryo abortion at early developmental stages. With the protocol developed here, microspores of 60–80% of donor plants could be induced to produce embryos, although embryo yields were low, i.e. 2–5 embryos per 10 buds. Some genotypes responded to culture conditions with high numbers of embryo formation (100–150 embryos per 10 buds) but most of these subsequently failed to mature. The pattern of cell division and morphological changes of the microspores in culture were studied using various microscopic techniques.     

Ethanol production by Zymomonas mobilis CP4 from sugar cane chips

Summary The fermentation of large sugar cane chips (1.0–1.5 in) to ethanol by Zymomonas mobilis CP4 (Z. mobilis) was studied in two glass fermentors operating with culture circulation for agitation (the EX-FERM type): a. A laboratory scale(2.5 liter) cylindrical vessel; b. A bench scale (8 liter) wide vessel. Z. mobilis cultures consumed 89–96% of the cane sucrose, converting it to ethanol by 90–97% of the theoretical yield in the laboratory scale fermentor and by 83–90% in the bench scale fermentor culture. Comparative Saccharomyces spp. cultures in laboratory fermentor consumed 96–98% of the cane sucrose, with ethanol conversion of only 75–79% of the theoretical yield.These preliminary results indicated that sucrose in agricultural size sugar cane chips was ethanol fermentable as compared to small size sugar cane chips or to sugar cane juice. Z. mobilis CP4 cultures converted sucrose more efficiently to ethanol than Saccharomyces spp. as shown in the laboratory scale fermentor studies.The ethanol yields in a wide bench scale fermentor cultures were slightly lower than in a laboratory fermentor.     

蝴蝶兰合子和胚的分离

该文用酶解一振荡法分离蝴蝶兰受精后的胚囊,然后显微解剖出合子、早期原胚及接近成熟的球形原胚。酶解液由O．7％-1．3％的纤维素酶、0．6％-1．0％果胶酶和10％甘露醇组成,pH5．8,酶解时间20-30min。分离的蝴蝶兰早期原胚和接近成熟的球形原胚均发现有发达的胚柄吸器。     

In vitro culture promotes partial autonomous endosperm development in unfertilized ovules of wild-type Arabidopsis thaliana var. Columbia

Partial endosperm development without paternal genome involvement was induced in unpollinated ovaries of wild-type Arabidopsis thaliana cultured in vitro. Unpollinated pistils were cultured on hormone-free Murashige and Skoog (MS) medium with addition of 6% sucrose and supplemented with: benzylaminopurine (BAP; 2 mg l^–1) combined with naphthylacetic acid (NAA; 0.1 mg l^–1), 2,4-dichlorophenoxyacetic acid (2,4-D; explants exposed to 1-h auxin shock 20 or 40 mg l^–1, and transferred to hormone-free MS medium). Initiation of autonomous endosperm (AE) development was induced on all media used in 54 ovules from 39 cultured ovaries (26%), with an average frequency of 1.4 ovules/ovary. The highest frequency of partial endosperm formation occurred on media combining the two growth regulators BAP and NAA (59% of ovaries had ovules with AE), although endosperm development was also induced on hormone-free medium (in 20.5% of ovaries). The number of AE nuclei ranged from 2 to ~50, depending on the day of culture and medium; neither cellularization nor differentiation on specific regions typical for endosperm of wild-type Arabidopsis, were noted. Fertilization independent endosperm most probably originated from the secondary nucleus, but involvement of the polar nuclei could not be excluded, as indicated by nuclear size and structure. In vitro conditions did not influence egg cell proliferation. Gynogenic embryos were observed neither in the ovules with autonomous endosperm nuclei nor in ovules without endosperm induction.     

Hyphal growth of Phytophtora cinnamomi on pine callus tissue

A procedure was developed which demonstrates the expression of differential resistance in pine callus tissues to the fungal pathogen Phytophthora cinnamomi Rands. Callus tissues were maintained on a modified Murashige and Skoog medium with 10^–5M 2,4-D and inoculated with hyphae of P. cinnamomi at 26°C in the dark. The number of intracellular hyphae was used as an index of resistance. Loblolly and loblolly × shortleaf pine hybrids were determined to be more resistant to infection and invasion by the fungus than were shortleaf and Virginia pine.Abbreviations (AL) loblolly pine—Alabama - (PL) South Carolina - (AS) shortleaf pine—Alabama - (CS) Georgia - (AV) Virginia pine—Alabama - (H1) loblolly × shortleaf pine hybrids—14–42 × 6-I-43 - (H2) I-523 × 6-D-8     

Regeneration of somatic embryos from protoplasts isolated from an embryogenic suspension culture of white spruce (Picea glauca)

Regeneration of white spruce (Picea glauca) somatic embryos from protoplasts derived from an embryogenic suspension culture was accomplished using a culture medium containing 2 mgl^–1 2,4-D and 1 mgl^–1 6-BAP. Divisions within 2 days led to plating efficiencies in the order of 24% after 9 days. A reduction in the osmoticum, necessary for sustained growth, was carried out gradually over 30 days. Embedding in agarose and culture in 5 cm petri dishes prior to transfer of agarose blocks to a bead type culture, led to the formation of somatic embryos as early as 23 days after isolation and yielded plating efficiencies in the order of 5–10% after 35 days culture.