Multiplication Of Somatic Embryos Research Articles

Plant Regeneration from Somatic Embryogenic Suspension Cultures of Date Palm.

Somatic embryogenesis is one of the most important technologies for plant regeneration of elite date palm cultivars. Recently, considerable progress has been made in the development and optimization of this technique from embryogenic cell suspension cultures. This chapter describes a procedure for the rapid development of a large number of somatic embryos from embryogenic cell suspension cultures. An efficient plant regeneration protocol via somatic embryogenesis from cell suspension cultures starting with shoot-tip explants to plantlet acclimatization also is fully described. Low concentrations of 6-benzylaminopurine (BAP) to 0.3mg/L and high rate of subcultures each 7 days lead to improve the establishment and multiplication of somatic embryos in suspension cultures by limiting oxidative browning, associated with high total phenols and peroxidase activities. The detailed morphological observations have revealed the cells destined to become somatic embryos. Activated charcoal (AC) at 0.15g/L has a positive effect on growth rate of somatic embryos by reducing tissue and medium browning, phenolics, and peroxidase activity.

Evaluation of biochemical markers during somatic embryogenesis in Silybum marianum L.

In present report effects of explants type, basal media and plant growth regulators (PGRs) were tested for induction of indirect somatic embryogenesis in medicinally important plant Silybum marianum L. Leaf, petiole and root explants were exploited in vitro on B5 (Gamborg), SH (Schenk and Hildebrandt) and MS (Murashige and Skoog) media for induction of embryogenic callus followed by somatic embryogenesis. Highest callus induction frequency (76 ± 4.8 %) was recorded when petiole explants of in vitro derived plantlets were cultured on B5 medium supplemented with 1.5 mg l−1 2,4-dichlorophenoxyacetic acid (2,4_D) in combination with 1.5 mg l−1 Thidiazuron (TDZ). Induction and multiplication of somatic embryos were observed, when the embryogenic calluses were sub-cultured on to B5 medium containing 0.5 mg l−1 2,4-D plus 1.5 mg l−1 TDZ. At this PGRs treatment, 77 % of the cultures responded with 39.1 somatic embryos per callus. Furthermore, MS0 medium was indicated more reponsive for growth and maturation of somatic embryos. Analysis of biochemical markers during various growth phases in somatic embryogenesis revealed that somatic embryos exhibited highest level of total carbohydrate, starch, ascorbic acid and total free amino acids. However, higher protein levels were detected in non-embryogenic callus. Nevertheless, considerable amount of silymarin (4.1 mg g−1 DW) was detected in somatic embryos than other growth phases. Thus, the present study concluded that biochemical and physiological changes during embryogenesis are influenced by interplay of explants type, basal media and PGRs.

The Effect of Gibberellin on Somatic Embryo Growth and Maturation and Plantlet Regeneration of Tangerine (Citrus reticulata Blanco.) var. Batu 55

The effect of gibberellin at multiplication stage on somatic embryo growth and maturation and plantlet regeneration of tangerine ( Citrus reticulata Blanco.) var. Batu 55 was assessed. Somatic embryo at globular phase was cultured on MT media + 30 gL -1 sucrose and various concentrations of gibberellin (0, 2, 4, 6, and 8 ppm). The somatic embryo was maturated on MT media + 500 ppm malt extract + 73 mM sorbitol + 73 mM galactose. Cotyledonary somatic embryo was regenerated into plantlet on MT media + 500 ppm malt extract + 30 gL -1 sucrose + 2 ppm GA 3 . The results showed that the addition of gibberellin in somatic embryo multiplication stage increased somatic embryo growth and maturation and plantlet regeneration of tangerine. Optimum concentration of gibberellin needed for somatic embryo growth was 4 ppm which yielded two-fold fresh weight compared to control. The percentage of maturation was very low below 5%. The addition of gibberellin in media at embryo multiplication stage slightly increased the percentage of maturation about 1-2%. Thirty percent of cotyledonary embryo was able to regenerate into plantlet. The addition of gibberellin in media at embryo multiplication stage increased the regeneration percentage, even the addition of 8 ppm gibberelline yielded regeneration percentage up to 70%. Keywords: gibberellin, growth, maturation, plantlet regeneration, somatic embryo

Secondary multiplication of somatic embryos in banana (Musa spp. AAA) in semisolid medium: effect of the culture vessel type and sterilization method

The objective of this work was to evaluate the influence of the type of culture vessel, and sterilization method on secondary multiplication of somatic embryos of the banana cultivar Grande naine (AAA) in semisolid culture medium. From 200 µl of embryogenic cell aggregates, somatic embryos in the globular stage were formed, which were used in the experiments to compare the types of culture vessel (glass and plastic) and the form of sterilization – humid heat (autoclave) and chemical (Vitrofural®). The evaluations of the number of somatic embryos that were multiplied were done at 15 and 20 days of culture. The results obtained with respect to the multiplication phase were highly promising, 40.25 and 80.15 ± 5.0 somatic embryos at 15 and 20 days of culture in glass vessel with plastic cover and using the autoclave as the sterilization method. The results presented in this work open the door for the use of this phase in the mass propagation protocol of this crop via somatic embryogenesis.

Histological analysis and transcription profiles on somatic embryogenesis in interspecific hybrids of <i>Elaeis guineensis × E. oleifera</i>

Elaeis guineensis (African oil palm) and E. oleifera (American oil palm) are bred to attain high oil yields, disease resistances, and decelerated shoot elongation. We cultivated immature zygotic embryos from backcrossed and F1 interspecific progenies on media containing 110, 150, or 200 mg·l-1 2,4-diclorophenoxyacetic acid (2,4-D) to obtain embryogenic cultures. These were set to multiply on medium containing 8 mg·l-1 2,4-D or lower concentrations of auxins and finally we induced plantlets regeneration, from each zygotic embryo, independently, in order to have the clones organized according to their respective origins. Reductions in auxins induced cultures to revert from highly embryogenic into competent for embryogenesis and finally to organogenic degenerated callus lines. Histology and the expression of SOMATIC EMBRYOGENESIS RECEPTOR KINASE, DEHYDRIN, DEFENSIN, TRANSPOSASE, and LIPID TRANSFER PROTEIN were analyzed on four callus lines representative of morphological aspects consistently observed. The highest number of embryogenic cultures was obtained on 150 mg·l-1 2,4-D. Maturation and multiplication of somatic embryos through secondary embryogenesis occurred simultaneously on 8 mg·l-1 2,4-D. LIPID TRANSFER PROTEIN expression was detected in one of the embryogenic cultures and correlated with protoderm onset. Three six-week cycles on induction medium yielded 1.5 shoots above 6 cm per poly-embryogenic complex, which performed better than embryoids individualized mechanically. Rooting was observed for 77% and 82% of shoots from these two types of explants, respectively. Rooted plantlets ready for acclimatization were obtained nine months after shoot induc

Somatic embryogenesis and bulblet regeneration in snakehead fritillary (Fritillaria meleagris L.)

Induction of in vitro morphogenesis of mature zygotic embryos of Fritillaria meleagrisL. was investigated. Somatic embryogenesis and whole plant regeneration were achieved. Isolated zygotic embryos were cultured on MS medium that contained 3% sucrose, 0.7% agar, 250.0 mg/l casein hydrolysate, 250.0 mg/l L-proline, 80 mg/l adenine sulfate and 1.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) or thidiazuron (TDZ). Embryogenic callus was derived from mature zygotic embryos after 4 weeks on TDZ containing medium. Somatic embryos at the early stages of development arose from the surface of the embryogenic callus. Multiplication of somatic embryos, formation of bulblets and shoot development were observed on the same medium. Scale sections prepared from the formed bulblets were cultured on MS media supplemented with 2,4-D (0.1 to 10.0 mg/l) and TDZ (0.05 to 2.0 mg/l). Somatic embryos and bulblets were frequently produced from the scale sections. This is a successful report of plant regeneration through somatic embryogenesis for this very important medicinal and horticultural plant. Histological observation revealed that plants of F. meleagris L. were regenerated via somatic embryogenesis. Key words: Bulbous plant, medicinal plant, snakehead fritillary, somatic embryos.

Low concentrations of BAP and high rate of subcultures improve the establishment and multiplication of somatic embryos in date palm suspension cultures by limiting oxidative browning associated with high levels of total phenols and peroxidase activities

Taking into account the recalcitrance of date palm ( Phoenix dactylifera L.) to tissue culture, establishment and proliferation of embryogenic suspension cultures in two date palm cultivars, namely Boufeggouss (BFG) and Bouskri (BSK) was implemented using liquid medium with different concentrations of the 6-benzylaminopurine (BAP) (0.3, 0.4, 0.5 mg/l) and various subculturing times (7 days, 15 days and 20 days). Total phenols and peroxidase activities have been determined and oxidative browning process checked in different cultures. The liquid medium with 0.3 mg/l BAP allowed cell suspension cultures to produce the greater globular embryos and the highest number of somatic embryos in comparison with the other treatment for BFG and BSK date palm cultivars. The low rate of culture transfers (every 15 or 20 days) tended to increase the phenolic contents and peroxidase activities in plant tissue leading to an enhancement of tissues/cells browning and then a decrease in the proliferation of embryogenic cells. The transfer of cultures on fresh medium every 7 days allowed a substantial reduction of tissue/cell oxidative browning, which is related to reduction of phenolic compounds and decrease in peroxidase activities with a resultant increase in the proliferation of embryogenic cells. This result suggests that oxidative browning is mainly peroxidase-based in date palm suspension cultures. Relationships between low concentrations of BAP as cytokinin, high growth of embryogenic cells, low contents of phenolics, decrease of peroxidase activities and oxidative browning process, are discussed, in order to optimize date palm multiplication through somatic embryogenesis and solve recalcitrance of many date palm cultivars to tissue culture.

IN VITRO REGENERATION OF ALLIUM AFLATUNENSE BY SOMATIC EMBRYOGENESIS

In vitro somatic embryogenesis and bulblet formation of Allium aflatunense in mature zygotic embryo culture were studied. Induction of morphogenesis was achieved on media supplemented with BDS mineral solution, 30 g L -1 sucrose, solidified by 0,7 mg L -1 agar and (in mg L -1 ): inositol 100.0, nicotinic acid 1.0, vitamin B 1 1.0, vitamin B 6 1.0, casein hydrolysate 100.0, L-proline 500.0, glicin 2.0, 2,4-D (2,4-dichlorophenoxyacetic acid, 2 mg L -1 ) or TDZ (thidiazuron, 2 mg L -1 ). Zygotic embryos cultured on BDS medium supplemented with 2,4-D formed friable embryogenic calli and some somatic embryos were formed. Somatic embryos were formed directly in the coleoptile and radicule region of zygotic embryos cultured on media with TDZ. The bulblet formation was also observed on BDS medium containing TDZ. Multiplication of somatic embryos, as well as, bulblets were observed after subsequent transfer on hormone free medium. Somatic embryos and bublets germinated after cold treatment (8 weeks) at +4°C.

Proliferation and maintenance of embryogenic capacity in elm embryogenic cultures

This paper investigates maintenance and proliferation of somatic embryogenesis systems for Ulmus minor and U. glabra. Proliferation occurred with subculture of embryogenic calluses. The calluses were mainly formed by friable nodules composed of meristematic cells organized into proembryogenic cell masses (PEMs) and thin-walled vacuolated parenchymatic cells. Cotyledonary embryos, with procambial strands and differentiation of their vascular tissues as well as visible root meristems, were identifiable after 18d of culture on a proliferation medium with 0.44 μM benzyladenine (BA). The shoot meristem was only occasionally well developed. Somatic embryo multiplication from elm embryogenic calluses is a clearly asynchronic system, and PEMs as well as embryos at all stages of development are observed simultaneously at the end of subculture period. Factors affecting the proliferation of elm embryogenic callus, such as culture medium, carbon source and genotype, were studied. Basal medium (MS) or medium supplemented with 0.44 μM BA produced the highest number of somatic embryos. Somatic embryo production was higher with sucrose or glucose than with maltose, and significant differences were also found among the four embryogenic lines tested. The use of liquid medium with filter paper support is an essential step for the survival of isolated somatic embryos during the germination stage. The addition of 0.22 μM BA′ to liquid MS medium was the best treatment for germination and plantlet conversion of elm somatic embryos.

Somatic embryogenesis of the banana hybrid cultivar FHIA-18 (AAAB) in liquid medium and scaled-up in a bioreactor

The development of somatic embryos in liquid culture medium has a number of advantages for large-scale propagation of plants. This paper describes an improved system for the mass propagation via somatic embryogenesis of the banana hybrid cultivar FHIA-18 (AAAB). Explants from immature male flowers were used to form high frequency embryogenic tissue, this tissue was then used to establish embryogenic cell suspensions in a basic MS medium plus 1.0 mg l−1 biotin, 100 mg l−1 glutamine, 100 mg l−1 malt extract (Sigma), 1.0 mg l−1 2,4-D and 45 g l−1 sucrose. Secondary multiplication of somatic embryos was achieved in liquid media in rotary shaker and in bioreactors. The number of embryos per litre obtained with 80.0% DO2 and effects of pH were also studied. A high regeneration percentage of plants were obtained (89.3%) in only 1 month of culture, somatic embryos were then placed to germinate in temporary immersion systems and field testing of somaclonal variation.