Gil S. Cruz

Somatic embryogenesis and plant regeneration from zygotic embryos of Feijoa sellowiana berg

Abstract Two distinct types of somatic embryos were induced from the cotyledonary region of callused zygotic embryos of Feijoa sellowiana cultured in MS medium supplemented with a wide range of 2,4-dichlorophenoxyacetic acid (2,4-D) concentrations (0.5–5.0 mg/l) singly or in combination with kinetin (Kn) (0.1–0.5 mg/l). Although less effective than 2,4-D, 1-napthaleneacetic acid (NAA) and 3-indolebutyric acid (IBA) could also induce somatic embryogenesis, whereas no embryos were observed if Kn alone was added to the culture medium. Only 5 days of treatment with 2,4-D were required to induce somatic embryogenesis. Longer exposures, until 14 days, increased the number of zygotic embryos responding positively to the treatment. The continued presence of 2,4-D in the culture medium did not prevent embryo development until the cotyledonary stage, but did not improve further the results. A great number of the formed somatic embryos carried different kinds of abnormalities which drastically reduced their germination capacity. Normal embryos germinated after being transferred to a modified Murashige and Skoogs (MS) medium containing gibberellic acid (GA 3 ) (0.5 mg/l) and kinetin (0.1 mg/l). Histological studies of embryogenic material showed that somatic embryos may arise directly from single cells. More often, however, quite extensive clumps or layers of meristematic cells are formed, mainly from dividing epidermal cells, from which numerous embryos are differentiated.

Somatic embryogenesis and plant regeneration in myrtle (Myrtaceae)

Somatic embryos of myrtle (Myrtus communis L.) were induced from mature zygotic embryos cultured in MS medium supplemented with several concentrations of 2,4-D (2.26 μM – 18.98 μM) or Picloram (2.07 μM – 16.5 μM) combined with 0.087 M or 0.23 M sucrose. For all the concentrations of 2,4-D or Picloram tested, 0.087 M sucrose proved to be more effective than 0.23 M. The best frequencies of induction were obtained in a medium containing 2.26 μM 2,4-D in which 97.3% of the explants produced somatic embryos. Although most embryos were produced from the adaxial side of the cotyledons, some of them differentiated from the hypocotyl. Secondary somatic embryos were often seen arising from the periphery of the former somatic embryos. Somatic embryo development was not synchronous but practically all the embryos germinated well after being transferred to media containing GA3 (0.29, 0.58 and 1.44 μM) alone. When benzyladenine was combined with gibberellic acid, germinating somatic embryos produced adventitious shoot buds which contributed to an increase in plantlet regeneration. Histological observations suggested that somatic embryos arise from the upper surface of the cotyledons probably from peripheral cells. Polyphenol-rich cells were usually seen in association with meristematic-like cells from which somatic embryos originate or with earlier steps of somatic embryo differentiation. Regenerated plants were phenotypically normal, showing a diploid (2n = 22) set of chromosomes.

Improvement of somatic embryogenesis inFeijoa sellowiana berg (Myrtaceae) by manipulation of culture media composition

SummarySomatic embryos could be induced from the cotyledons of zygotic embryos from immature fruits ofFeijoa sellowiana Berg (Feijoa) in the presence of a wide range of concentrations of fructose, glucose, maltose, and sucrose. Mannitol or sorbitol alone were ineffective. The highest frequencies of induction (99%) and the greatest number of somatic embryos per explant (134) were obtained with 0.4M fructose and 0.3M sucrose, respectively. This sucrose concentration also showed greater induction capacity than equimolar combinations of its monosaccharide constituents combined. Somatic embryo development was arrested at the globular stage at concentrations higher than 0.5M of all the sugars tested. When transferred to solid germination medium containing 2.0 mg/liter (5.77µM) gibberellic acid, 0.5 mg/liter (2.32µM) kinetin, and 0.029M sucrose, somatic embryos formed under 0.3 or 0.4M sucrose had better germination capacity than those induced under lower (0.1 and 0.2M) concentrations, as assessed by the frequency of explants presenting germinated embryos and by the number of plants obtained from those explants. On liquid media of similar composition somatic embryos did not germinate. Our data suggest that high (0.3 to 0.4M) carbohydrate levels improve somatic embryogenesis by acting both as carbon source and as osmotic regulator.

Somatic embryogenesis and plant regeneration in carob (Ceratonia siliqua L.)

SummarySomatic embryos of carob (Ceratonia siliqua L.) were induced from cotyledonary segments excised from immature seeds when cultured on Murashige and Skoog media supplemented with several combinations of 6-benzylaminopurine (BA) and indole-3-butyric acid (IBA). The best frequencies of induction (33.8%) were obtained when 4.4 μM BA and 0.5 μM IBA were used. Shoots were also sporadically formed in the same media. When IBA was replaced by other auxins in the induction media, only α-naphthaleneacetic acid (NAA) and indole-3-acetic acid (IAA) could induce somatic embryogenesis, although at lower rates than IBA. 2,4-Dichlorophenoxyacetic acid and 4-amino-3,5,6-trichloropicolinic acid were completely ineffective. Besides culture media composition, the developmental stage of the explants at the time of culture showed a strong influence on somatic embryogenesis induction, with cotyledons from stage II pods providing the highest levels of induction. By contrast, the genotype of the explant did not determine a significant role in the induction process. Attempts to achieve somatic embryo germination were mostly unsuccessful, since only shoot development was observed; the highest frequencies of development occurred on media containing only gibberellic acid (3.0 μM). For plant regeneration, the developed shoots were further rooted on IBA-supplemented media, and the plantlets obtained were transferred to soil, where c. 88% of them survived. Histological observations showed the presence of morphologically normal and abnormal somatic embryos, the latter displaying an abnormal pattern of vascular bundles. Ultrastructural analysis showed that the cells of the globular embryos had a dense cytoplasm, whereas those not involved in somatic embryo formation showed signs of senescence. Histological studies were also used to distinguish between somatic embryos and shoots originated in the same media.

Somatic embryogenesis in myrtaceous plants

The Myrtaceae is a large family that includes about 3.000 species distributed by over 100 genera (Heywood, 1993; Paiva, 1997). Most species are concentrated in the South hemisphere, specially in tropical and subtropical regions of Australia, South and Central America and Southeast Asia (Heywood, 1993). A few species can be found in Southern Europe and North Africa, surrounding the Mediterranean sea. Eugenia and Eucalyptus are the genera including the highest number of species, whereas Myrtus communis is the only species native to Europe. Some species of Eucalyptus (e.g. E. citriodora, E. globulus) were introduced in several regions of Europe (Paiva, 1997). Meanwhile, they became the dominant species in some areas of Portugal and Spain (Mcvaugh, 1963).

Somatic embryogenesis and plant regeneration in Cyphomandra betacea (Cav.) Sendt

Callus cultures with globular proembryogenic structures were induced from zygotic embryos and hypocotyl segments of Cyphomandra betacea on MS medium supplemented with 2,4-D. Proembryogenic structures produced somatic embryos and plantlets on regulator-free basal medium. Pieces of embryogenic callus subcultured on medium with the same original composition gave rise to new globular structures and the potential for plantlet regeneration has been maintained for over a year. The histological examination of these proembryogenic structures suggested that somatic embryos arise from single cells. Regenerated plants are phenotypically normal, having diploid chromosome numbers (2n = 24).

Somatic Embryogenesis Induction in Tamarillo (Cyphomandra betacea)

Cyphomandra betacea (Cay.) Sendtn. usually known as tamarillo or tree tomato, is a solanaceous soft wood tree (Fig. 1A) grown for their edible fruits (Fig. 1B). The species, which may reach 2 – 4 meter height (Slack, 1976), produces tomato-like red, orange or yellow fruits according with the cultivars. The fruits of the red cultivar are the most popular due to their more striking appearance and better flavour (Slack, 1976). They are generally 2 – 3 inches long and 2 inches in diameter possessing many seeds (Hooker, 1899). Also included in the same genus are the species Casana (Cyphomandra casana), Mountain Tomato (C. crassifolia) and Guava Tamarillo (C. fragans). The plant can be propagated by seeds or cuttings (Fougue, 1973) or it may be grafted in Solanum mauritianum (Slack, 1976). In the first case, plants do not usually come true-to-type rendering difficult the propagation of selected genotypes (Barghchi, 1998). Several authors (see section 1.3) have also reported protocols for in vitro regeneration. The first pinkish flowers appear in spring, while the mature fruits are collected from October to April (Guimaraes et al., 1996). However, flower appearance and fruit maturity can be changed by pruning (Slack, 1976).

Somatic Embryogenesis Induction in Bay Laurel (Laurus nobilis L.)

Bay laurel or sweet bay (Laurus nohilis L.) is a member of the Lauraceae family, which includes about 2.500 species originating, predominantly, from tropical and subtropical regions with a few native to temperate climates (Heywood, 1993). Some of these species (e.g. Persea americana, Cinnamomum cantphora, Sassafras sp., Ocotea sp., Litsea sp.) are important sources of fruits, chemicals (oils) and timber. The genus Laurus comprises the species L. azorica which is confined to the Canary, Azores and Madeira islands, and L. nobilis which spread around the Mediterranean basin and is found in some areas of Portugal (Tutin, 1964). In Roman times, bay laurel was a symbol of glory and knowledge, being used to crown poets and heroes (Lanzara et al., 1978), a tradition kept for many centuries afterwards.

In vitro induction of haploid, diploid and triploid plantlets by anther culture of Iochroma warscewiczii Regel

Pollen of Iochroma warscewiczii Regel (Solanaceae) produced embryogenic calli or embryos inside anthers cultured on Nitsch & Nitsch medium. Two distinct pathways could be recognized in this process, one involving mainly the vegetative cell, and the second starting with two equal cells in the pollen grains.In all media tested, androgenesis initiation was highest when anthers contained pollen at the first mitosis, or close to it, at inoculation. High sucrose (7%) and calcium (11.3 mM) concentrations were found to be highly desirable for the induction of androgenesis in this species. Addition of benzylaminopurine (0.5 mg l−1) to the culture medium seems to slightly improve callus or embryo production. When all three factors were present at optimal concentrations as much as 13.9% of inoculated anthers were found to be embryogenic.Plantlet development from pollen embryos required lower sucrose (3%) and a combination of 0.1 mg l−1 benzylaminopurine and 0.5 mg l−1 gibberellic acid in the culture medium. Cytological analysis of 55 regenerated plantlets showed that about 49% were haploids, but diploid (ca. 49%) and triploid (ca. 2%) plants were also obtained.

Induction of pollen callus in anther cultures of Feijoa sellowiana Berg. (Myrtaceae)

SummaryAnthers of Feijoa sellowiana Berg. (feijoa) produced pollen callus when cultured in Murashige and Skoog medium containing 2,4-dichlorophenoxyacetic acid and benzyladenine or in nurse cultures. Somatic callus was also formed in large amounts from the connective and from the cut end of the filament. Anthers containing microspores at the stage immediately prior to the first pollen mitosis cultured in the presence of 3% sucrose, presented the highest frequencies of induction. Androgenetic divisions were initiated by the formation of two morphologically equal cells, the so-called B-pathway. Attempts to regenerate pollen plants were unsuccessful but leaf-like structures could be obtained in regeneration media containing combinations of gibberellic acid and benzyladenine.