Direct and Indirect Somatic Embryogenesis Induction in Camellia oleifera Abel

Camellia oleifera Abel. is an important woody oil species; however, the shortage of rapid and industrialized seedling culture is a large constraint on the development of the tea oil industry. Somatic embryogenesis (SE) is one of the main powerful biotechnological tools for plant mass regeneration, but the largely unknown SE in C. oleifera limits the scale production of clonal plants. In this study, we described a high-efficiency SE system via direct and indirect pathways in C. oleifera and investigated the effect of genotype, explant age and phytohormones on SE. In the direct pathway, somatic embryos were highly induced from immature seeds 220 days after full blossom, and the development of embryoids was achieved with a combination of 0.19 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.05 mg/L thidiazuron (TDZ). In the indirect pathway, embryogenic calli were induced from the same explants in medium containing 1.5 mg/L 2,4-D, while 0.75 mg/L 2,4-D treatment led to high proliferation rates for embryogenic calli. The addition of 0.19 mg/L 2,4-D alone stimulated the production of globular embryos while causing a 75% loss of the induction rate in the heart embryo stage. Upon transfer of the globular embryos to phytohormone-free medium, an optimal induction rate of 62.37% from globular embryos to cotyledonary embryos was obtained. These data suggest that the subsequent differentiation process after the globular embryo stage in ISE is more similar to an endogenous phytohormones-driven process. Mature embryos germinated to produce intact plantlets on half-strength MS basal medium with a regeneration rate of 63.67%. Histological analysis confirmed the vascular bundle isolation of embryoids from the mother tissue. We further studied the different varieties and found that there were no significant genotype differences for SE induction efficiency in C. oleifera. Thus, we established a high-efficiency induction system for direct and indirect somatic embryogenesis (ISE) in C. oleifera and regenerated intact plantlets via SE, not organogenesis. ISE has a more complicated induction and regulatory mechanism than direct somatic embryogenesis. The improved protocol of SE would benefit mass propagation and genetic manipulation in C. oleifera.

Induction of nucellar embryogenesis in nucellar tissue of Citrus aurantifolia for their clonal multiplication

Citrus aurantifolia (lime) has been selected as explant for nucellar embryogenesis. Nucellus is a non-vascularized tissue being true-to-type same as mother plant, meristematic cells have no plasmodesmata connection, no virus can pass through nucellus, thus it seems to be a good material for production of virus freeplantlet.Putrescine at 0.25 or 0.5 mg1-1 and anapthaleneacetic acid at 0.10 mg1-1 supplemented to nutrient formulation were most effective in alleviating cotyledonary proliferation and fasciation while promoting embryo-to-embryo proliferation producing numerous whitish globular embryos were formed. For further development of globular embryos to well-differentiated cotyledonary embryos, additional presence of 2-isopentenyladenine at concentrations of 0.10 or 0.25 mg 1-1 was essential, contrary to incorporation of 0.10 or 0.25 mg 1-1 6benzylaminopurine, which promoted excessive proliferation of cotyledonary structures and their fasciation while zeatin at the same concentrations produced intermediate response. In the optimum treatment containing 0.25 mg l-1 putrescine, 0.10 mg 1-1 isopentenyladenine, 0.10 mg 1-1 indole-3-acetic acid and 100 mg l-1 malt extract, an average 10 well-developed embryos per culture were formed, besides some abnormal cotyledonary structures. Well-developed embryos measuring ca. 2 cm. in length (leaving the root) germinated 100% into plantlets, during 60 days, in the additional presence of amino acid supplement comprising, 5 mg 1-1 each of L-arginine, L-asparagine, L-histidine, L-cysteine, L-lysine and 10 mg l-1 L-glutamine. Such plantlets nurtured in a different medium attained a height of ca. 4 cm in 45 days before they were taken out for ex vitro growth. There was 100% transplant success and the plants grew normally.

Pollination, Fertilization, and Embryo Development in Southern China Fresh-eating Jujube

To explore the reasons for seed abortion in southern China fresh-eating jujube, improve its reproductive biology, and provide a theoretical basis for the crossbreeding of jujube, we carried out self-pollination and cross-pollination experiments with Ziziphus jujuba Mill. ‘Zhongqiusucui’ as the female parent. We observed the process of pollen tube growth in pistil and embryo development by fluorescence microscopy and paraffin section methods. The results show there were self- and cross-incompatibilities during pollination and fertilization, and there were no significant differences in pollen germination and pollen tube growth between self-pollination and cross-pollination. It took at least 4 hours for pollen and stigma to recognize each other, 6 hours for pollen to germinate on the stigma, and 12 hours for the pollen tube to penetrate the mastoid cells of the stigma. After 48 hours of pollination, the pollen tube reached one third of the style. The pollen tube remained stagnant 72 to 120 hours after pollination, and remained at one third of the stylar canal. Simultaneously, the pollen tubes on the stigma twisted and interacted with each other, and expanded into a spherical shape. A few pollen tubes reached the ovary and completed fertilization. However, some early globular embryos degenerated before forming into globular embryos and resulted in the formation of empty embryo sacs, which leads to seed abortion. In conclusion, the poor pollination and fertilization, and the blocked development of the embryo resulted in seed abortion in Z. jujuba ‘Zhongqiusucui’.

Pollination, Fertilization, and Embryo Development in Southern China Fresh-eating Jujube

To explore the reasons for seed abortion in southern China fresh-eating jujube, improve its reproductive biology, and provide a theoretical basis for the crossbreeding of jujube, we carried out self-pollination and cross-pollination experiments with Ziziphus jujuba Mill. ‘Zhongqiusucui’ as the female parent. We observed the process of pollen tube growth in pistil and embryo development by fluorescence microscopy and paraffin section methods. The results show there were self- and cross-incompatibilities during pollination and fertilization, and there were no significant differences in pollen germination and pollen tube growth between self-pollination and cross-pollination. It took at least 4 hours for pollen and stigma to recognize each other, 6 hours for pollen to germinate on the stigma, and 12 hours for the pollen tube to penetrate the mastoid cells of the stigma. After 48 hours of pollination, the pollen tube reached one third of the style. The pollen tube remained stagnant 72 to 120 hours after pollination, and remained at one third of the stylar canal. Simultaneously, the pollen tubes on the stigma twisted and interacted with each other, and expanded into a spherical shape. A few pollen tubes reached the ovary and completed fertilization. However, some early globular embryos degenerated before forming into globular embryos and resulted in the formation of empty embryo sacs, which leads to seed abortion. In conclusion, the poor pollination and fertilization, and the blocked development of the embryo resulted in seed abortion in Z. jujuba ‘Zhongqiusucui’.

MORPHOLOGICAL CHANGES DURING THE DEVELOPMENT OF SOMATIC EMBRYOS OF SAGO (Metroxylon sagu Rottb.)

Development of somatic embryos of sago (Metroxylon sagu Rottb.) on agar-solidified medium are highly varied producing heterogeneous seedlings. Understanding of this phenomenon may help in improving the cultural procedures and conditions of sago<br />somatic embryogenesis to obtain uniform seedlings in a large scale. This experiment was conducted at the laboratory for plant cell culture and micropropagation, Indonesian Biotechnology Research Institute for Estate Crops from January to March 2006 to examine morphological changes i.e. color and development stages of sago during their somatic embryo development on an agar-solidified medium. Twenty single globular somatic embryos of sago with specific color (yellowish, greenish, and reddish) were cultured in a Petri dish supplemented with a solid medium. The medium was a micronutrients-modified MS (MMS) with half strength of macronutrients containing 0.01 mg l-1 ABA, 2 mg l-1 kinetin, 20 g l-1 sucrose, 0.5 g l-1 activated charcoal, and 2 g l-1 gelrite. Parameter observed was the percentage of embryo’s number based on color and developmental stage. The result showed that at the end of 6-week culture passage, most originally greenish (80.8%) and reddish (95.8%) embryos remained unchanged in their colors, whereas almost half of the originally yellowish embryos turned to greenish and only 30%<br />remained yellowish. At the same time, single globular embryos have changed gradually into the next developmental stages, although not all of the embryos were germinated. The initial color of embryo affected the rate of the developmental stage changes. Yellowish and greenish globular embryos developed more rapidly into cotyledon or germinant stages at 58% and 55% respectively, in 6 weeks than the reddish ones (41%). Therefore, the yellowish and greenish embryos are the best sources of material for in vitro mass propagation and synthetic seed production of sago.

MORPHOLOGICAL CHANGES DURING THE DEVELOPMENT OF SOMATIC EMBRYOS OF SAGO (Metroxylon sagu Rottb.)

Development of somatic embryos of sago (Metroxylon sagu Rottb.) on agar-solidified medium are highly varied producing heterogeneous seedlings. Understanding of this phenomenon may help in improving the cultural procedures and conditions of sago<br />somatic embryogenesis to obtain uniform seedlings in a large scale. This experiment was conducted at the laboratory for plant cell culture and micropropagation, Indonesian Biotechnology Research Institute for Estate Crops from January to March 2006 to examine morphological changes i.e. color and development stages of sago during their somatic embryo development on an agar-solidified medium. Twenty single globular somatic embryos of sago with specific color (yellowish, greenish, and reddish) were cultured in a Petri dish supplemented with a solid medium. The medium was a micronutrients-modified MS (MMS) with half strength of macronutrients containing 0.01 mg l-1 ABA, 2 mg l-1 kinetin, 20 g l-1 sucrose, 0.5 g l-1 activated charcoal, and 2 g l-1 gelrite. Parameter observed was the percentage of embryo’s number based on color and developmental stage. The result showed that at the end of 6-week culture passage, most originally greenish (80.8%) and reddish (95.8%) embryos remained unchanged in their colors, whereas almost half of the originally yellowish embryos turned to greenish and only 30%<br />remained yellowish. At the same time, single globular embryos have changed gradually into the next developmental stages, although not all of the embryos were germinated. The initial color of embryo affected the rate of the developmental stage changes. Yellowish and greenish globular embryos developed more rapidly into cotyledon or germinant stages at 58% and 55% respectively, in 6 weeks than the reddish ones (41%). Therefore, the yellowish and greenish embryos are the best sources of material for in vitro mass propagation and synthetic seed production of sago.

Embryological studies of reciprocal crosses between Vicia faba and Vicia narbonensis

A study was undertaken to asses the reciprocal crossability between <em>Vicia faba</em> and <em>Vicia narbonensis</em>. Flower buds or only ovaries of several varietes and genotypes were cross-pollinated in vivo (green house and field) and in vitro. Only few pollen tubes passed the style and entered into the ovary. On the whole number of 5320 cross pollinated in vivo and in vitro flowers and ovaries of <em>Vicia narbonensis</em> only 78 globular hybrid embryos were observed. After cross pollination in vivo of 3860 flower buds and ovaries of <em>Vicia faba</em> globular embryos developed in 124 ovules. The highest number of globular embryos were obtained when the <em>Vicia faba</em> line 1/33 was pollinated with <em>Vicia narbonensis</em> lines P3, P5, 150, SE.Embryogenesis proceeded till the 6-10 day after pollination, however, karyological disturbances in the cells of embryos and endosperm were often noticed at earlier stages. In vitro pollen grains of <em>Vicia faba</em> germinated on stigmas and ovaries of <em>Vicia narbonensis</em>, a significant increase in the growth of pollen tubes was noticed after ovary pollination. The technique of in vitro pollination was not suitable for <em>Vicia faba</em> as the inoculated explants died shortly after transferring onto the medium. The results indicate that finding a more suitable genotype for crossing may give a chance to obtain higher number of embryos (example line 1/33) - thus sufficient number for culturing them on media.

chlorophyll autofluorescence in globular and heart-shaped embryos of some dicotyledons

The regions in early embryos of several species display chlorophyll autofluorescence in a certain order. First, autofluorescence in <em>Pisum sativum</em> appears in the basal part of globular embryos; in <em>Lathyrus vernus</em> in the basal part of early heart embryos; in <em>Cardamine pratensis</em> at the sides of the hypocotyl or in <em>Phaseolus vulgaris</em> in the hypocotyl of elongating heart-shaped embryos. Chlorophyll autofluorescence in an embryo proper of <em>Pisum</em> coincides with the development of a lamellar system in the plastids. The suspensorial plastids remain undifferentiated with one or two DNA positive nucleoids. <em>Cardamine</em>, <em>Lathyrus</em>, <em>Phaseolus</em> and <em>Pisum</em> suspensors give no chlorophyll autofluorescence.

In vitro placental self and cross pollination in some species

Excised placentae with ovules of <i>Primula pubescens</i>, <i>P. auricula</i>, <i>Scopolia carniolica</i>, <i>Digitalis purpurea</i>, <i>Torenia fournieri</i> and <i>Chionodoxa luciliae</i> were self pollinated in vitro and the development of seeds was observed. The same method was used for obtaining hybrid globular embryos from crosses between: <i>P. pubescens</i> x <i>P. auricula</i>, <i>Scopolia carniolica</i> x <i>Physochlaina praealta</i>, <i>Melandrium album</i> x <i>Silene saxifraga</i> and <i>M. album</i> x <i>Arenaria pungens</i>.

Early somatic embryogenesis in Heliconia chartacea Lane ex Barreiros cv. Sexy Pink ovary section explants

The present work evaluated the development of embryogenic callus from transversal ovary sections. The experiments were carried out under two experimental regimes using combinations of IAA (0; 5.71; 8.56; 11.42; 14.27μM) and 2,4-D (0; 13.57; 18.10; 22.62μM) or combinations of 2,4-D with BA (0; 4.43; 6.65; 8.87; 11.09μM). Assessments were made of anatomical aspects of the callus and for the presence of embryogenic structures using cytochemical and histological analyses and stereomicroscopic and scanning electronic microscopic observations. Treatments with 2,4-D and IAA produced friable calluses demonstrating cellular acquisition of morphogenetic competence as well as the formation of pro-embryogenic sectors. The expression of embryogenic program could be observed, with proembryogenic cell clusters developing into globular embryos. These results offer the possibility of using new types of explants for culturing helicons that avoid the growth of endophytic bacteria.