Somatic Embryogenesis In Rosa Chinensis CV. ‘Parson’s Pink China’

This study investigated the effects of explant type, plant growth regulator concentration, callis status, medium conversion time, and medium tilt on the growth of rose somatic embryos. The results showed that Rosa chinensis cv. ‘Parson’s Pink China’ leaves could induce normal embryogenic calli but petioles could not. When the 2,4-dichlorophenoxyacetic acid concentration was 3.0 mg/L, the callis induction rate was the highest in the embryo proliferation medium (EP medium) supplemented with 0.5mg/L kinetin, and white and reddish-brown translucent calli were the main type of embryogenic calli induced. As the culture time in EP medium was extended, the relative induction rate for secondary embryos and multicotyledon secondary embryos gradually increased when transferred to embryo maturation medium (EM medium), but the induction rate for somatic embryos decreased. Placing the EM medium at an angle of 45° made the somatic embryos germinate faster and the germination rate was also higher. The germination buds produced by the somatic embryos with two cotyledons showed the fastest germination and greatest survival rates. The results of this experiment will help improve somatic embryo regeneration rates and explore new ways of regeneration, and lays the foundation for further optimization of the somatic embryo genetic transformation system.

Three in one: evolution of viviparity, coenocytic placenta and polyembryony in cyclostome bryozoans

Background Placentation has evolved multiple times among both chordates and invertebrates. Although they are structurally less complex, invertebrate placentae are much more diverse in their origin, development and position. Aquatic colonial suspension-feeders from the phylum Bryozoa acquired placental analogues multiple times, representing an outstanding example of their structural diversity and evolution. Among them, the clade Cyclostomata is the only one in which placentation is associated with viviparity and polyembryony—a unique combination not present in any other invertebrate group. Results The histological and ultrastructural study of the sexual polymorphic zooids (gonozooids) in two cyclostome species, Crisia eburnea and Crisiella producta, revealed embryos embedded in a placental analogue (nutritive tissue) with a unique structure—comprising coenocytes and solitary cells—previously unknown in animals. Coenocytes originate via nuclear multiplication and cytoplasmic growth among the cells surrounding the early embryo. This process also affects cells of the membranous sac, which initially serves as a hydrostatic system but later becomes main part of the placenta. The nutritive tissue is both highly dynamic, permanently rearranging its structure, and highly integrated with its coenocytic ‘elements’ being interconnected via cytoplasmic bridges and various cell contacts. This tissue shows evidence of both nutrient synthesis and transport (bidirectional transcytosis), supporting the enclosed multiple progeny. Growing primary embryo produces secondary embryos (via fission) that develop into larvae; both the secondary embyos and larvae show signs of endocytosis. Interzooidal communication pores are occupied by 1‒2 specialized pore-cells probably involved in the transport of nutrients between zooids. Conclusions Cyclostome nutritive tissue is currently the only known example of a coenocytic placental analogue, although syncytial ‘elements’ could potentially be formed in them too. Structurally and functionally (but not developmentally) the nutritive tissue can be compared with the syncytial placental analogues of certain invertebrates and chordates. Evolution of the cyclostome placenta, involving transformation of the hydrostatic apparatus (membranous sac) and change of its function to embryonic nourishment, is an example of exaptation that is rather widespread among matrotrophic bryozoans. We speculate that the acquisition of a highly advanced placenta providing massive nourishment might support the evolution of polyembryony in cyclostomes. In turn, massive and continuous embryonic production led to the evolution of enlarged incubating polymorphic gonozooids hosting multiple progeny.

Primary and secondary somatic embryogenesis in Jatropha curcas L. From leaf transverse thin cell layers

An efficient method for plant regeneration in Jatropha curcas L. via primary and secondary somatic embryogenesis culture from ex vitro leaves of 6-month-old plants was presented in this study. Leaves were cut into transverse thin cell layers (tTCLs) and cultured on MS medium supplemented with kinetin (KIN) at 0.5, 1.0, 1.5, and 2.0 mg/l in combination with indole-3-butyric acid (IBA) at 0.1, 0.5, and 1.0 mg/l or 2,4-dichlorophenoxyacetic acid (2,4-D) at 1.0, 1.5 and 2.0 mg/l . The highest embryogenic callus formation rate (89.3%) was obtained on medium supplemented with 1.0 mg/l KIN and 1.5 mg/l 2,4-D. The calli were selected for the study of primary somatic embryogenesis on MS medium containing 2,4-D (0.01, 0.03, 0.05, and 0.07 mg/l) or KIN (0.5, 1.0, 1.5, and 2.0 mg/l). The highest primary somatic embryos formation rate (76.67%) was achieved on MS medium supplemented with 1.0 mg/l KIN. The primary embryos were cultured on medium supplemented with KIN (0.1, 0.5, 1.0, 1.5, and 2.0 mg/l) combined with 0.2 mg/l indole-3-butyric acid (IBA) or 0.05 mg/l 2,4-D. The combination of 1.5 mg/l KIN and 0.05 mg/l 2,4-D was suitable for secondary embryos formation. Embryos proliferated rapidly, and the highest number of secondary embryos (77.5 embryos) wasobtained from a single primary embryos inoculated. Results also showed that the addition of proline (0.75 g/l) or spermidine (0.15 mM) to the culture medium increased the number of secondary embryos considerably. The fully developed plantlets exhibiting healthy roots and shoots were obtained when somatic embryos were sub-cultured onto B5 medium containing 1.5 mg/l IBA.

Keragaman morfologi selama perkembangan embrio somatik sagu (Metroxylon sagu Rottb.) Morphological variations during the development of somatic embryos of sago (Metroxylon sagu Rottb.)

Summary In vitro culture of sago (Metroxylon sagu Rottb.) on an agar-solidified medium consists of somatic embryos of different sizes, colors, and developmental stages. One gram of mostly globular somatic embryos were cultured on a solid medium to observe their morphological variations with respect to embryo size, color, and developmental stage over one passage of six weeks culture. The medium was a modified-MS medium with half-strength of macronutrients containing 0.01 mg/L ABA and 2 mg/L kinetin. At the end of culture passage, fresh weight of embryo increased by 2.3 folds. The embryo numbers increased by more than two times indicating the formation of secondary embryos. The average size of sago somatic embryos did not change significantly over the culture period; however, the embryo size was already highly varied at the start and increased gradually as the embryo developed. At the initial of culture, 33.7 % of the embryos were yellowish, 64.1 % were greenish, and 2.2% were reddish. By the end of the culture the composition of yellowish embryos increased to 51.2 %, greenish embryo decreased to 42.5 % and red embryos increased to 6.3 %. At the initial culture, 61 % of the embryos were at the globular, 9 % at heart-shape and 30 % at torpedo stage. Generally globular embryos developed into later-stage embryos as the culture progressed, although almost 56% of the embryos remained at the globular stage after the sixth week.Ringkasan Kultur in vitro sagu (Metroxylon sagu Rottb.) pada medium padat terdiri dari embrio somatik dalam berbagai ukuran, warna, dan fase perkembangan. Satu gram embrio somatik yang sebagian besar dalam fase globuler dikulturkan pada medium padat untuk mengamati keragaman morfologi embrio dalam hal ukuran, warna dan fase perkembangan dalam satu periode kultur enam minggu. Medium kultur adalah MS modifikasi dengan setengah hara makro serta penambahan zat pengatur tumbuh ABA 0,01 mg/L dan kinetin 2 mg/L. Pada akhir masa kultur bobot embrio segar meningkat 2,3 kali dibandingkan awal masa kultur. Jumlah embrio juga mengalami peningkatan sebesar lebih dari dua kali yang menunjukkan adanya pembentukan embrio somatik sekunder. Ukuran rata-rata embrio tidak berubah secara signifikan selama masa kultur akan tetapi ukuran embrio telah sangat beragam pada awal kultur dan terus meningkat hingga akhir kultur. Warna embrio mengalami perubahan selama periode kultur. Pada awal kultur dijumpai 33,7 % embrio berwarna kuning, 64,1 % embrio hijau, dan 2,2 % embrio merah. Pada akhir kultur presentase embrio kuning meningkat menjadi 51,2 %, embrio hijau menjadi 42,5 %, dan embrio merah 6,3 %. Pada awal kultur, dijumpai 61 % embrio pada fase globuler, 9 % fase bentuk-hati dan 30 % fase torpedo. Umumnya embrio globuler berkembang menjadi embrio fase lanjut selama kultur berlangsung, namun 56 % embrio masih tetap dalam fase globuler pada minggu keenam.

Keragaman morfologi selama perkembangan embrio somatik sagu (Metroxylon sagu Rottb.) Morphological variations during the development of somatic embryos of sago (Metroxylon sagu Rottb.)

Summary In vitro culture of sago (Metroxylon sagu Rottb.) on an agar-solidified medium consists of somatic embryos of different sizes, colors, and developmental stages. One gram of mostly globular somatic embryos were cultured on a solid medium to observe their morphological variations with respect to embryo size, color, and developmental stage over one passage of six weeks culture. The medium was a modified-MS medium with half-strength of macronutrients containing 0.01 mg/L ABA and 2 mg/L kinetin. At the end of culture passage, fresh weight of embryo increased by 2.3 folds. The embryo numbers increased by more than two times indicating the formation of secondary embryos. The average size of sago somatic embryos did not change significantly over the culture period; however, the embryo size was already highly varied at the start and increased gradually as the embryo developed. At the initial of culture, 33.7 % of the embryos were yellowish, 64.1 % were greenish, and 2.2% were reddish. By the end of the culture the composition of yellowish embryos increased to 51.2 %, greenish embryo decreased to 42.5 % and red embryos increased to 6.3 %. At the initial culture, 61 % of the embryos were at the globular, 9 % at heart-shape and 30 % at torpedo stage. Generally globular embryos developed into later-stage embryos as the culture progressed, although almost 56% of the embryos remained at the globular stage after the sixth week.Ringkasan Kultur in vitro sagu (Metroxylon sagu Rottb.) pada medium padat terdiri dari embrio somatik dalam berbagai ukuran, warna, dan fase perkembangan. Satu gram embrio somatik yang sebagian besar dalam fase globuler dikulturkan pada medium padat untuk mengamati keragaman morfologi embrio dalam hal ukuran, warna dan fase perkembangan dalam satu periode kultur enam minggu. Medium kultur adalah MS modifikasi dengan setengah hara makro serta penambahan zat pengatur tumbuh ABA 0,01 mg/L dan kinetin 2 mg/L. Pada akhir masa kultur bobot embrio segar meningkat 2,3 kali dibandingkan awal masa kultur. Jumlah embrio juga mengalami peningkatan sebesar lebih dari dua kali yang menunjukkan adanya pembentukan embrio somatik sekunder. Ukuran rata-rata embrio tidak berubah secara signifikan selama masa kultur akan tetapi ukuran embrio telah sangat beragam pada awal kultur dan terus meningkat hingga akhir kultur. Warna embrio mengalami perubahan selama periode kultur. Pada awal kultur dijumpai 33,7 % embrio berwarna kuning, 64,1 % embrio hijau, dan 2,2 % embrio merah. Pada akhir kultur presentase embrio kuning meningkat menjadi 51,2 %, embrio hijau menjadi 42,5 %, dan embrio merah 6,3 %. Pada awal kultur, dijumpai 61 % embrio pada fase globuler, 9 % fase bentuk-hati dan 30 % fase torpedo. Umumnya embrio globuler berkembang menjadi embrio fase lanjut selama kultur berlangsung, namun 56 % embrio masih tetap dalam fase globuler pada minggu keenam.

Manifestation of embryogenic potential in culture of zygotic embryos of Quercus robur L.

For the initiation of somatic embryogenesis early cotyledonary stage of zygotic embryo explants (from 15th July until late August) was suitable. The highest frequency of differentiation of somatic embryos was obtained on cotyledons of zygotic embryos cultured on basal modified medium MS (with 1/2 concentration macronutrients) or WPM medium containing 500 mg•l^-1 glutamine, proline and casein hydrolysate and supplemented with 2,4-D (1,0-2,0 mg•l^-1) and BAP (0,5-1,0 mg•l^-1). The development of somatic embryos was direct and indirect and the process was continuous over a long period. Primary somatic embryos were able to produce secondary embryos. Repetitive somatic embryogenesis led to the proliferation of a large number of new somatic embryos on their cotyledons, hypocotyl or radicula. The process of embryo differentation is asynchronous - various stages of somatic embryos could be observed in embryogenic culture. A somatic embryo conversion was rare on tested media. Embryo germination occured on medium containing BAP (0,1 mg•l^-1) or on medium with ABA and GA₃ (each 0,2 mg•l^-1) after a previous culture on WPM medium without plant growth regulators supplemented with sorbitol (6%). The embryo germination occurred also on WPM medium with 0.2 mg•l^-1 BAP when cultures were mantained at 2^oC for 4 weeks. Only 8 somatic embryos developed into plantlets. Their transplantation to <em>in vivo</em> conditions was unsuccessful.

Plant regeneration via somatic embryogenesis from immature leaves in Tetrapleura tetraptera (Schum. & Thonn.) Taub.

Plant regeneration via somatic embryogenesis was assessed using immature leaf, petiole and apical meristem explants in Tetrapleura tetraptera. Somatic embryos were induced in the immature leaf using MS basal medium supplemented with 2,4-D and matured on MS basal medium containing BAP. Medium supplemented with 12 mg/l 2,4-D had the highest (43.1%) percentage of embryogenic calluses from immature leaf explants. Conversion of embryogenic callus to mature primary somatic embryo occurred in the medium that contained 1.2 mg/l BAP. Development of secondary embryogenic calluses to matured secondary embryos was highest (98.0%) in the medium with 0.4 mg/l BAP, while the highest average number of mature secondary embryos (6.0) was obtained in the same medium. Medium supplemented with 1.0 mg/l BAP and 0.5 mg/l IBA had the highest (38.7%) percentage of explants with shootbuds. The highest (18.1%) percentage of shoot elongation was obtained in medium with 1.0 mg/l BAP and 20 mg/l IBA. Shootbuds survived and produced roots on medium free of plant growth regulators. Shoots obtained on medium supplemented with 1.0 mg/l BAP and 20 mg/g IBA recorded the highest number of roots per plantlet (7.5) with no apparent morphological abnormality.

Induction of somatic embryogenesis in immature seeds of guavatree cv. Paluma

The biotechnological techniques may help solve many problems of guava culture, such as the high perishability of fruits. Somatic embryogenesis can generate highly multiplicative cell cultures and with high regenerative potential, serving as basis for genetic transformation. The aim of this work was to obtain somatic embryogenesis of guava (Psidium guajava L.) cv. Paluma. Immature seeds were used, and they were inoculated in MS environment containing 400 mg L-1 of L-glutamine, 100 mg L-1 myo-inositol, 60 g L-1 sucrose, 100 mg L-1 ascorbic acid and supplemented with different types and concentrations of growth regulators. Embryogenic callus appeared after 37 days of culture in an environment containing 1.0 mg L-1 2.4-D + 2.0 mg L-1 2-ip, in 7% of the explants. After 65 days of culture, the treatment containing 0.5 mg L-1 CPA showed 20% of explants with direct embryos, while the treatment with 1 mg L-1 had 14% of explants with direct embryos and 7% of explants with embryogenic callus. In 66.6% of embryos regenerated with 0.5 mg L¹ CPA there was the formation of secondary embryos. The use of IASP and BAP, aiming embryogenesis proliferation, led to an increase in the cellular proliferation, but calli apparently lost their embryogenic potential.