Angela Carra

Somatic embryogenesis and plant regeneration from pistil transverse thin cell layers of lemon (Citrus limon)

Abstract Callus induction, somatic embryogenesis and plant regeneration were obtained in Citrus limon (L.) Burm. (cv. Femminello) from cultures of pistil transverse thin cell layer explants [(t)TCLs]. Explants were cultured on two different media, based on Murashige and Skoog salts and vitamins, supplemented with 500 mg l−1 malt extract (MSI), or 500 mg l−1 malt extract and 13.3 μM 6-benzylaminopurine (MSII). Sucrose (146 mM) was used as carbon source. Somatic embryos appeared 3 months after culture initiation from stigma and style (t)TCLs; they were observed at the surface of the (t)TCL-derived callus. Although ovary (t)TCLs showed the highest callus formation, they never differentiated somatic embryos. Percentages of embryo formation from (t)TCLs incubated on MSI (13% and 2% for stigma and style, respectively) were lower than those from (t)TCLs incubated on MSII (36% and 7% for stigma and style, respectively). The embryogenic response of stigma (t)TCLs was usually higher than that of style (t)TCLs. After about 3 months, somatic embryos developed into plantlets at high frequencies (57% and 62% for stigma- and style-derived somatic embryos, respectively).

Regeneration of Algerian Citrus germplasm by stigma/style somatic embryogenesis

Stigma/style somatic embryogenesis is one of the efficient methods in plant regeneration of most Citrus ssp., without inducing somaclonal variations. Furthermore, somatic embryogenesis from style/stigma proved to be effective in the elimination of the main citrus virus and virus-like diseases. This technique was applied on Algerian citrus collection. Different Citrus species [ Citrus sinensis (L.) Osbeck, C. limon (L.) Burm, C. reticulata Blanco, C. paradisi Macfad, C. reshni Hort. ex Tan., C. jambhiri Lush and C. maxima (Burm.) Merrill] were chosen and tested for the presence of the main virus and virus-like agents. Most of the genotypes showed to be infected, mainly by viroid agents. Closed flowers were collected and in vitro cultured on a Murashige and Skoog (MS) medium supplemented with 6- benzylaminopurine. All explants produced callus about 4 to 9 days after culture initiation, whereas embryogenesis occurred after 38 to 150 days in most of the cultured genotypes. Formed embryos were cultured in a single tube before in vivo acclimatization. After sanitary assays, regenerated plants were shown to be free from the agents detected in the mother trees. Key words : Algeria, citrus germplasm, plant regeneration, sanitation, somatic embryogenesis.

Somatic embryogenesis of muskmelon ( Cucumis melo L.) and genetic stability assessment of regenerants using flow cytometry and ISSR markers

A new protocol for in vitro regeneration through direct somatic embryogenesis for two muskmelon cultivars (Cucumis melo L., “Mashhadi” and “Eivanaki”) is reported. Somatic embryos were obtained culturing 4- and 8-day-old cotyledons, seeds, and hypocotyls on Murashige and Skoog medium supplemented with three different hormonal combinations never tested so far for melon (naphthoxyacetic acid (NOA) + thidiazuron (TDZ), NOA + 6-banzylaminopurine (BAP), and 2,4-dichlorophenoxyacetic acid (2,4-D) + N-(2-chloro-4-pyridyl)-N′-phenylurea (4-CPPU)). Results were compared with those obtained when explants were cultivated in the presence of 2,4-D + BAP, previously used on melon. Embryogenesis occurred more successfully in 4-day-old cotyledons and seeds than hypocotyls and 8-day-old cotyledons. The best result was achieved with NOA + BAP. Genotypes significantly affected embryogenesis. The number of embryos in “Eivanaki” was significantly higher than that in “Mashhadi.” Embryo proliferation when explants were maintained in jars (9.3%) was found to be higher compared to that in petri dishes. For the first time, genetic stability of regenerated melon plants was evaluated using inter-simple sequence repeat markers. Polymerase chain reaction (PCR) products demonstrated a total of 102 well-resolved bands, and regenerants were 93% similar compared to the mother plant. Somaclonal changes during embryogenesis were evaluated by flow cytometry, showing 91% of the same patterns in regenerated plants. The results suggest that the new hormone components are effective when applied for in vitro embryogenesis of muskmelon as they show a high frequency in regeneration and genetic homogeneity.

Efficient plant regeneration via somatic embryogenesis in bulbing fennel using immature flower explants

In order to optimize plant regeneration techniques for bulbing fennel [Foeniculum vulgareMill. subsp. vulgare var. azoricum (Mill.) Thell] via somatic embryogenesis, three different media were tested on four Sicilian fennel ecotypes derived from seed collections. Plant regeneration was obtained through the use of immature inflorescences as explants. Through comparison of different auxins, naphthoxyacetic acid resulted in high-level induction of embryogenic callus at the lowest concentration (4 μM), in combination with 6-benzylaminopurine or thidiazuron. After germination of somatic embryos, regenerated plantlets were recovered on medium containing half-strength Murashige and Skoog salts and vitamins in the absence of plant growth regulators. Regenerated plants were successfully acclimatized (70 %) in a growth chamber prior to transplantation into an open field. The plants appeared phenotypically normal, and both genetic stability and uniformity were confirmed by random amplified polymorphism DNA analysis. The results represent a significant advance on previous reports, due to the high embryogenic ability of immature inflorescence from different ecotypes and the high percentage of regenerated plantlets acclimatized in ex vitro conditions.

In vitro propagation of the relict laperinne’s olive (Olea europaea L. subsp. Laperrinei)

Abstract Olea europaea L. subsp. laperrinei (Oleaceae) is an endemic taxon of the mountainous regions of central Sahara, consisting of currently fragmented and small relict populations. The tree can propagate vegetatively or by seed, but no recent natural regeneration was observed in the Algerian massive populations, some of which are considered threatened with extinction. Sterile triploid individuals were also identified in some populations showing increasing vigour. As a result of its long persistence and despite its rarity, the Laperrine’s olive is an iconic component of Saharan mountain ecosystems. The aim of this study is to develop an efficient micropropagation protocol for both diploid and triploid Laperrine’s olive to safeguard and preserve this genetic resource. Best shoot propagation was obtained on a modified Murashige and Skoog medium supplemented with 9.2 μM zeatin. Best rooting rate of regenerated shoots was achieved on the same culture medium supplemented with 4.8 μM indole-3-butyric acid. In absence of morphological changes of in vitro regenerated plants acclimatized to the greenhouse, genetic conformity was assessed by simple sequence repeat screening. Our results suggest that in vitro propagation could be a useful tool for conservation of both diploid and triploid threatened Laperrine’s olive.