Secondary Embryogenesis Research Articles

Cyclic Somatic Embryogenesis in Indonesian Elite Theobroma cacao L. Clones

To date, in vitro somatic embryogenesis is the only option for the mass vegetative propagation of cocoa. The somatic embryogenesis of Indonesian cocoa clones SUL1 and SUL2 was investigated, focusing on primary and cyclic secondary embryogenesis. The study showed that staminode explants were more effective than petal explants in generating primary somatic embryos (SEs), especially when cultured in liquid medium containing 2 mg/L 2,4-D and 0.25 mg/L kinetin, with the staminodes of SUL2 producing a significant number of globular SEs. In contrast, SUL1 showed limited SE production. The study also demonstrated that fragmenting cotyledons and hypocotyls of the SEs of SUL2 and culturing them on an induction medium supplemented with 2,4,5-T and proline resulted in a high yield of secondary SEs. This cyclic embryogenesis process, in which the SEs remained attached to the maternal tissue, facilitated continuous SE production and development. The addition of proline was found to improve the quality of SEs, leading to higher production of well-organized, milky SEs with a better-defined meristematic structure. These results suggest a promising protocol to produce SEs from cocoa, with implications for plant transformation and gene editing applications.

Recurrent secondary embryogenesis in androgenic embryos and clonal fidelity assessment of haploid plants of Tea, Camellia assamica ssp. assamica and Camellia assamica ssp. lasiocaylx

Globular androgenic haploid embryos of TV21 and TV19 cultivars of Camellia ssp., obtained on embryo induction medium (EIM), Murashige and Skoog medium with 10 µM 6-benzylaminopurine (BAP), 3 µM gibberellic acid (GA3), 800 mg l−1 L-glutamine and 200 mg l−1 L-serine, were pretreated with 18 µM abscisic acid (ABA) and 25 g l−1 mannitol for 20 days in liquid medium to attain maturation. The pre-treatment favoured the primary globular embryos to grow and pass through all the developmental stages from globular, heart-shape, torpedo shape to fully matured docotyledonous embryos. Fivefold and threefold increase in secondary embryogenesis (SE) was attained in Camellia assamica ssp. assamica (TV21; Assam Type) and Camellia assamica ssp. lasiocaylx (TV19; Cambod Type), respectively, when pre-treated primary embryos were transferred to the liquid embryo development (ED) medium, supplemented with tenfold reduced concentration of growth regulators that were present in EIM. Embryos were germinated well on 10 µM BAP, 0.5 µM GA3, 1 µM indole-3-butyric acid (IBA), 80 mg l−1 L-glutamine and 20 mgl−1 L-serine, following liquid-to-semi-solid transition method. Well developed shoots and roots were formed when embryos were initially kept for four weeks in liquid germination medium, followed by their transfer to semi-solid medium on similar medium composition. The method promoted recurrent secondary embryogenesis in lesser time with highest germination rate, 66.6% and 30.3% in TV21 and TV19 cultivars, respectively. Histology and field emission scanning electron microscopy (FESEM) were performed to determine ontological events during embryo development. Clonal fidelity of haploid plantlets was assessed using random amplified polymorphic DNA (RAPD) to ensure no somaclonal variations occurred during the multistep process of secondary embryogenesis, embryo maturation and germination. A two-step transition protocol from liquid-semi-solid medium is beneficial for embryos to overcome hyperhydricity. Secondary embryogenesis is the best mode to obtain genetically uniform true-to-type haploid plantlets.

ВЛИЯНИЕ УСЛОВИЙ КУЛЬТИВИРОВАНИЯ НА ЧАСТОТУ ПРОРАСТАНИЯ/РЕГЕНЕРАЦИИ МИКРОСПОРОГЕННЫХ ЭМБРИОИДОВ BRASSICA OLERACEA L.

Most genotypes of white cabbage (Brassica oleracea L.) have a low frequency of microspore-derived embryos germination and regeneration through adventitious shoots or secondary embryogenesis. Increasing the frequency of regeneration and the formation of seedlings can ensure the production of a large number of doubled haploids. This work studied the effects of gelling agents, such as agar (11 g/l), agargel (4.5 g/l), and phytagel (2 g/l), on the frequency of regeneration/germination of microspore-derived embryos of white cabbage (B. oleracea L.), as well as the effect of a cold incubation of kohlrabi cabbage embryos (B. oleracea var. gongylodes L.) at 5°C for 3, 6, 9, 12 and 15 days in complete darkness. The studies showed that when culturing embryos on regeneration medium B5 solidified with agar, the embryo germination and seedling regeneration were higher than those on media solidified with agargel and phytagel. No embryo germination but vitrification of embryo tissues was observed on media containing agargel and phytagel. Cold incubation of Kohlrabi cabbage embryos at 5°C for 3, 6 and 9 days in complete darkness twice increased the frequency of embryo germination compared to the control sample. More extended periods of cold incubation for 12 days in the dark, on the other hand, twice reduced the rate of direct embryo germination compared to the control sample, and for 15 days – 6 times.

Maturation and Conversion of Somatic Embryos Derived from Seeds of Olive (Olea europaea L.) cv. Dahbia: Occurrence of Secondary Embryogenesis and Adventitious Bud Formation.

Maturation and conversion of somatic embryos are two crucial steps that hamper the development of efficient somatic embryogenesis systems in olive. Herein, a simple and efficient protocol for the maturation and conversion of olive somatic embryos is reported. Globular somatic embryos derived from seeds of cv. Dahbia were cultured on either half-strength olive (OM) or olive cyclic embryogenesis (ECO) media, with and without plant growth regulators (PGRs). The embryos reached the cotyledonary stage in 9 weeks, but those cultured on ECO medium containing 0.1 mg·L−1 6-(dimethylallylamino)purine (2iP), 0.1 mg·L−1 6-benzyladenine (BA) and 0.05 mg·L−1 indole-3-butyric acid (IBA) exhibited the largest sizes, with an average of 4.7 mm. Somatic embryo conversion into plantlets was evaluated using different culture media (half-strength OM or one-third strength Murashige and Skoog (MS)), light conditions (light or dark) and desiccation pretreatments. The highest rate of somatic embryo conversion (45%) was observed under a 16 h photoperiod on half strength OM medium containing 0.1 mg·L−1 gibberellic acid (GA3) and 0.1 mg·L−1 1-naphthalene acetic acid (NAA). The embryos that failed to germinate showed either necrosis, cotyledon greening with no further conversion, adventitious bud formation or secondary embryogenesis. The findings of this study will be beneficial for biotechnological applications in olive.

Enhanced Secondary Somatic Embryogenesis in Suspension Culture of Four Diploid Banana Cultivars from Kerala

ABSTRACT Secondary somatic embryogenesis was observed in the suspension culture of bract-derived banana calli in the presence of liquid Murashige and Skoog medium with additives such as glutamine (0.68–34.21 µM), biotin (0.40–20.46 µM), or ascorbic acid (0.56–28.38 µM) along with malt extract (100 mg/l). After the initiation of homogenous cell suspension, fully developed somatic embryos were observed after fifth subculture in the four cultivars. Somatic embryos were successfully regenerated in basal MS medium and were transplanted after fifth week. After fifth subculture, the frequency of somatic embryos increased in each culture flask. Maximum number of somatic embryos were observed in cv. Sannachenkadali (46.12 ± 0.85a) when cultured in liquid MS medium supplemented with biotin (8.18 µM) along with malt extract (100 mg/l), in followed by cv. Matti (44.75 ± 1.19a) having (AA) genome. SEM analysis revealed the presence of secondary somatic embryos on the surface of primary somatic embryos in each culture and it attributed a high frequency of somatic embryogenesis. The addition of biotin along with malt extract increased the frequency of secondary somatic embryogenesis in the diploid banana cultivars.

Enhanced Secondary Somatic Embryogenesis in Suspension Culture of Four Diploid Banana Cultivars from Kerala

ABSTRACT Secondary somatic embryogenesis was observed in the suspension culture of bract-derived banana calli in the presence of liquid Murashige and Skoog medium with additives such as glutamine (0.68–34.21 µM), biotin (0.40–20.46 µM), or ascorbic acid (0.56–28.38 µM) along with malt extract (100 mg/l). After the initiation of homogenous cell suspension, fully developed somatic embryos were observed after 5th subculture in the four cultivars. Somatic embryos were successfully regenerated in basal MS medium and were transplanted after fifth week. After 5th subculture, the frequency of somatic embryos increased in each culture flask. Maximum number of somatic embryos were observed in cv. Sannachenkadali (46.12 ± 0.85a) when cultured in liquid MS medium supplemented with biotin (8.18 µM) along with malt extract (100 mg/l), in followed by cv. Matti (44.75 ± 1.19a) having (AA) genome. SEM analysis revealed the presence of secondary somatic embryos on the surface of primary somatic embryos in each culture and it attributed high frequency of somatic embryogenesis. Addition of biotin along with malt extract increased the frequency of secondary somatic embryogenesis in the diploid banana cultivars.

Effects of Genotype and Culture Conditions on Microspore Embryogenesis and Plant Regeneration in Brassica Rapa ssp. Rapa L.

Turnip is a biennial crop and, consequently, the creation of pure lines for breeding is a time-consuming process. The production of pure turnip lines using doubled haploids produced in isolated microspore culture has not been sufficiently developed. The aim of the present work was to determine some key factors inducing embryogenesis in the isolated microspore culture of turnip, as well as investigating the manners of embryo development. It was shown that the acidity of the medium is an important factor in embryo production; different optimal pH levels ranging from 6.2 to 6.6 corresponded to individual genotypes. Such factors as the cold treatment of buds and the addition of activated charcoal to the nutrient medium increased the responsiveness of all genotypes studied. The turnip variety ‘Ronde witte roodkop herfst’ demonstrated a genetic disorder in the development of microspores; namely, non-separation of some microspores from tetrads. In the in vitro culture, each of the daughter microspores developed on its own. This indicates the dependence of the possibility of embryogenesis in the turnip microspore culture on the genotype. Results suggest that the initiation of secondary embryogenesis in primary embryos leads to an increase in the proportion of doubled haploid plants.

Regeneration of transgenic plants by Agrobacterium-mediated transformation of Quercus ilex L. somatic embryos with the gene CsTL1

Here we describe for the first time a protocol for the genetic transformation of the holm oak Quercus ilex. Holm oak populations are seriously affected by a disorder known as oak decline syndrome. However, tolerant plants can be obtained by overexpression of pathogenesis-related (PR) proteins. The aim of the present study was to define a protocol for transforming somatic embryos (SEs) of holm oak with the gene CsTL1 encoding a chestnut thaumatin-like protein (PR protein). Lethal concentrations of kanamycin (kan) were determined in previous studies, in which SE formation was found to be inhibited by kan concentrations of 100 mg/L and above. Genetic transformation was only achieved when target explants were pre-cultured for one or 2 weeks, with a transformation efficiency of 2%. Transformation was also affected by genotype and was only successful in 2 of the 3 embryogenic lines evaluated. A total of 11 transformed lines (10 corresponding to line Q8 and 1 to line Q10–16) were obtained, all of which were maintained by secondary embryogenesis on proliferation medium (Schenk and Hildebrandt medium lacking plant growth regulators). The transgenic embryogenic lines were successfully cryopreserved by a vitrification-based procedure, thus enabling the lines to be preserved while tolerance tests were performed. The presence of CsTL1 in genomic DNA was confirmed by PCR, and expression of the gene was determined by qPCR. CsTL1 expression was up to 5.93 times higher in transgenic lines than in the corresponding untransformed line. Transgenic plants were produced from transformed SEs, with embryo conversion rates ranging from 2.8 to 66.7%.

Morphological and histological analysis of anther-derived embryos of coconut (Cocos nucifera L.)

Doubled-haploids are a great source of material for heterozygous coconuts to shorten the time taken for crop improvement through hybridization programs. However, low frequency of embryo conversion and formation of weak plantlets are the major limitations in the protocol. In the present study anther-derived embryos were analysed at cellular level through the histological observations to understand the occurrence of shoot differentiation in those embryos. Attempts were also made to optimise the 6-Benzylaminopurine (BAP) concentration in the regeneration medium. Among the tested BAP levels (5, 15, 25, 35, 45, 55 μM), the regeneration medium containing 25 and 35 μM gave rise to 51.5% sprouted embryos against the control that gave only 22.7%. Among the analysed anther-derived embryos sequential events of differentiation including the formation of provascular strands followed by vascular bundle, differentiation in to the growing point, giving rise to the secondary embryos and polarization in to the shoot and root pole were identified. The blunt embryos devoid any morphological sign of sprouting showed different cellular arrangement viz. haustorial structure without any meristematic point (47%), bipolar with shoot and root meristems (8%) and unipolar with either of the pole (45%). Thick haustorial cover lead to the physical dormancy inhibiting further development of the polar structures. The germinating embryos containing a single shoot gave rise to the healthy plants where as double, multiple or fused shoots formed the week plantlets. The findings can be used for further optimisation of the protocol to achieve a greater plant regeneration frequency. 1. Culture media supplemented with increased levels of 6-Benzylaminopurine (BAP) enhanced the regeneration frequency of anther-derived embryos. 2. Thick haustorial tissue created an obstacle effect on further development of the differentiated shoot apical meristem. 3. Formation of clustered plants through secondary embryogenesis or shoot multiplication at the early stage of plant regeneration caused to give rise to the week plants.

Embryogenic callus induction and fatty acid composition analysis of oil palm (Elaeis guineensis cv. Tenera)

Embryogenic callus (EC) induction and plantlet regeneration of oil palm (Elaeis guineensis) are essential platforms for the propagation of uniform hybrid cultivars in large-scale cultivation. In this paper, an embryogenic callus was successfully induced from leaf tissue from the ‘Tenera’ varieties of oil palm and regenerated into plantlets via secondary embryogenesis. The total lipid and separated triacylglycerol (TAG) contents of the EC were determined using GC–MS. The results showed that the EC had a total lipid content of approximately 9.46%, while TAG accounted for 32% of the total lipid content. Considering that the TAG content was more than 95% of the total fatty acids (FAs) in the mesocarp of ripe oil palm fruit, the FA profile of the TAG content in the EC was compared with the total lipid content in the mesocarp of oil palm. The results indicated that the FA profile of the TAG content in the EC was similar to that in the mesocarp, even though there were still significant differences in the content of individual FAs. The similar FA composition in the EC and mesocarp of oil palm will be a valuable marker for predicting FA profiles in the mesocarp of the mature transgenic oil palm fruit obtained from transformation and embryogenesis.

Somatic embryogenesis in Carica papaya as affected by auxins and explants, and morphoanatomical-related aspects.

The aim of this study was to evaluate somatic embryogenesis in juvenile explants of the THB papaya cultivar. Apical shoots and cotyledonary leaves were inoculated in an induction medium composed of different concentrations of 2,4-D (6, 9, 12, 15 and 18 µM) or 4-CPA (19, 22, 25, 28 and 31 µM). The embryogenic calluses were transferred to a maturation medium for 30 days. Histological analysis were done during the induction and scanning electron microscopy after maturing. For both types of auxin, embryogenesis was achieved at higher frequencies with cotyledonary leaves incubated in induction medium than with apical shoots; except for callogenesis. The early-stage embryos (e.g., globular or heart-shape) predominated. Among the auxins, best results were observed in cotyledonary leaves induced with 4-CPA (25 µM). Histological analyses of the cotyledonary leaf-derived calluses confirmed that the somatic embryos (SEs) formed from parenchyma cells, predominantly differentiated via indirect and multicellular origin and infrequently via synchronized embryogenesis. The secondary embryogenesis was observed during induction and maturation phases in papaya THB cultivar. The combination of ABA (0.5 µM) and AC (15 g L-1) in maturation medium resulted in the highest somatic embryogenesis induction frequency (70 SEs callus-1) and the lowest percentage of early germination (4%).

Origin of Secondary Somatic Embryos and Genetic Stability of the Regenerated Plants in Hevea brasiliensis

Micropropagation by secondary embryogenesis had been established in Hevea brasiliensis. However, origin and development of secondary embryos and genetic stability of the regenerated plants remained unclear. Visual and cytological observations found secondary embryos mainly arose from epidermal cells of cotyledons and might originate from single cells. The number of pro-embryos (45.00) peaked after 20 days of cultivation for cell differentiation. At the maturation STAGE, the sum of immature embryos including globular, heart-Like, and torpedo-liked secondary embryos (22.50) was approximately equal to the number of cotyledonary embryos (21.75), which was much lower than that of pro-embryos (45.00), indicating a great number of pro-embryos were not able to convert into cotyledonary embryos under the current culture conditions. Evaluation of the genetic stability through chromosome number counting and EST-SSRs fingerprint, showed the chromosome number of the regenerated plants was similar to the mother tree (2n=36), the variation rates of EST-SSRs loci and the regenerated plants initially increased and then decreased with multiplication cycles, and the variation rate of EST-SSRs loci was considered low (<2.61%), indicating the genome remained stable during multiplications. These results could further improve secondary embryogenesis system and inquire into its potential applications in H. brasiliensis.

Abnormal Somatic Embryo Reduction and Recycling in Grapevine Regeneration

The purpose of the study was to increase plant conversion efficiency and reduce waste of the abnormal somatic embryos; factors influencing both the embryo morphology in germination and the embryogenic callus reinduction rate in secondary embryogenesis were studied in Vitis vinifera L. ‘Thompson Seedless.’ Embryos having mono-, di-, and multiple-normal cotyledons, trumpet-shaped, fascicular, and fused abnormal cotyledons were observed during germination. Normal embryos of one, two, and multiple cotyledons have a relatively higher conversion rate (48.89–64.44%) than abnormal ones (2.22–22.22%). Histomorphology analysis of these embryos showed that normal somatic embryos had a better developed vasculature and shoot meristem than abnormal individuals. A lower percentage of abnormal embryos formed when they germinated on Murashige and Skoog’s medium (MS) and Woody Plant Medium (WPM) both of which contained 15 g/l sucrose and no plant growth regulators. Of all the abnormal embryos, only fused cotyledon embryos were suitable for recycling, and they were reinduced into the embryogenic callus. Cotyledons showed a higher reinduction rate (72.22%) than the other two parts: hypocotyls (4.44%) and roots (0%). In addition, recycled abnormal embryos produced many morphogenic types and this secondary embryogenesis could be used for embryo proliferation.

Somatic embryogenesis and regeneration of five multipurpose cassava landraces extensively integrated in African cropping system

ABSTRACTSuccessful development and adoption of transgenic cassava (Manihot esculenta Crantz) varieties in Africa depend on in vitro regeneration of landraces because of their agronomic value and amenability to genetic modification. The study investigated somatic embryogenesis from axillary bud and immature leaf-lobe explant and regeneration via shoot organogenesis in five cassava landraces. Landraces exhibited significant (P < 0.05) differences in frequencies of primary somatic embryo production, number of embryos per explant, and frequency of secondary embryos. The frequency of primary somatic embryogenesis ranged from 7.8 to 14.5%, whereas the number of embryos per explant varied from 5.3 to 10.6. However, only frequency of primary somatic embryos and number of embryos per explant showed significant (P < 0.05) differences when immature leaf lobe was used as explant. The frequency of primary somatic embryogenesis ranged from 42.7 to 49.2%, whereas number of embryos per immature leaf-lobe explant varied from 9.5 to 15.2 per explant. Secondary embryogenesis was cultivar-independent in the case of immature leaf-lobe explant. Cyclic and green (mature) embryogenesis showed no significant (P < 0.05) landrace differences in both axillary bud and immature leaf-lobe explants. Shoot regeneration from cotyledon of somatic embryos had significant (P < 0.05) landrace differences. The mean shoot-bud formation frequency of axillary bud and immature leaf-lobe explants was 51.4% and 37.2%, respectively. Root formation was efficient, with greater than 70% of shoots forming root in all landraces. Similarly, landrace differences were detected for the survival of acclimatized regenerated plants, with a mean of 94.7% for both explants. In conclusion, somatic embryos were produced from immature leaf and axillary bud explants of the landraces and the embryos were converted to plantlets via shoot organogenesis.

Coconut Micropropagation in Mexico using Plumule and Floral Explants

This paper focuses on the research efforts carried out by CICY in Mexico on micropropagation of coconut. They started during the nineties in collaboration with Wye College (UK) and ORSTOM-CIRAD (France), with the development of a protocol that was reproducible and more efficient than previous ones, based on plumule explants grown in different media based on Y3 medium added with activated charcoal, gelling agent and of particular importance growth regulators 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (BAP). Within the next decade basic research was carried out to study the process of somatic embryogenesis from plumule explants, with an approach including morpho-histological, physiological, biochemical and molecular points of view, in order to gain knowledge that could be useful to further improvement of the process. Also different practical approaches were tested including changes in the media formulation, embryogenic callus multiplication and secondary somatic embryogenesis. As a result a highly efficient protocol was developed that could potentially yield over a hundred thousand somatic embryos from a single plumule explant. Embryos were able to germinate and convert to plantlets, that after planting, successfully grew to sexual maturity and fruit production. This protocol is currently being scaled up to a semi-commercial level. Also within the past five years, a protocol using rachilla explants has been developed for the production of embryogenic callus and its multiplication, and embryos produced were able to germinate and convert to plantlets, setting the basis to develop a process for massive propagation of coconut, such as the one already developed using plumule explants.

Agrobacterium-mediated transformation of European chestnut somatic embryos with a Castanea sativa (Mill.) endochitinase gene

Chestnut blight, caused by Cryphonectria parasitica, is a severe disease that has devastated chestnut stands in North America and Europe. Genes encoding hydrolytic enzymes such as chitinases, which can degrade fungal cell wall components, are attractive candidates for improving disease resistance. This report describes a reliable and efficient protocol for the Agrobacterium-mediated transformation of somatic embryos of European chestnut with the endogenous CsCh3 gene that codes for chitinase. The transformation efficiency, determined on the basis of the fluorescence of surviving explants, was genotype-dependent. Although somatic embryos of all three lines evaluated were transformed, the best results were obtained with somatic embryos derived from line CI-9 (20 %). The addition of silver thiosulphate (20 or 40 μM) improved the transformation efficiency of somatic embryos derived from lines CI-3 and CI-9, although the differences were not significant. A total of 88 independent transformed lines were obtained. The presence of transgenes was confirmed by green fluorescent protein (GFP) expression, PCR and Southern blot analysis. Transgenic lines were maintained by secondary embryogenesis or cryopreservation following vitrification procedures. Maturation and germination of transformed somatic embryos yielded transgenic plants. Fluorescence indicating overexpression of the transgenes was observed in somatic embryos and also in shoots and leaves. No phenotypic differences were found relative to control plants, suggesting a lack of any cytotoxic effects of the GFP.

&lt;i&gt;In Vitro&lt;/i&gt; Secondary Embryogenesis Derived from Meta-Topoline Treatment on Mass Propagation of &lt;i&gt;Phalaenopsis&lt;/i&gt; ‘AMP 17’

Phalaenopsis ‘AMP 17’ is an important orchid commodity in Indonesia with high market demand; however, scaling up the orchid commercially is constrained by the availability and sustainability of qualified seedlings. To overcome the problem, a reliable in vitro propagation protocol, especially via secondary embryogenesis, was undertaken. In the present study, in vitro secondary embryogenesis derived from meta-topoline (mT) treatment on mass propagation of Phalaenopsis ‘AMP 17’ was successfully established. Embryos, as explant sources, were prepared by culturing meristem tips of flower stalk shoots on Murashige and Skoog (MS) medium containing 1.5 mg/L thidiazuron (TDZ) and 0.25 mg/L N6-benzylaminopurine (BAP) for ± 3 months. High secondary somatic embryo (SSE) formation up to 64.90% with 12.30 SSEs regenerated per embryo was determined on half-strength MS augmented with 0.5 mg/L BAP and 2.5 mg/L mT. The combination also stimulated the result of high multiplication rate of SSE formation, up to 10.1 fold on the third subculture, maintained low conversion rate of germinated-embryos down to 55% and improved qualified-growth of the germinated embryos. The mT treatment produced 86% survival plantlets with high qualified-performance. The system could be applied as an alternative method to step forward towards an improved propagation protocol, commercially efficient due to high productivity. Detail findings in each step were discussed.

Somatic Embryogenesis and Genetic Modification of Vitis.

Grapevine embryogenic cultures are ideal target tissues for inserting desired traits of interest and improving existing cultivars via precision breeding (PB). PB is a new approach that, like conventional breeding, utilizes only DNA fragments obtained from sexually compatible grapevine plants. Embryogenic culture induction occurs by placing leaves or stamens and pistils on induction medium with a dark/light photoperiod cycle for 12-16 weeks. Resulting cultures produce sectors of embryogenic and non-embryogenic callus, which can be identified on the basis of callus morphology and color. Somatic embryo development occurs following transfer of embryogenic callus to development medium and cultures can be maintained for extended periods of time by transfer of the proliferating proembryonic masses to fresh medium at 4-6-week intervals. To demonstrate plant recovery via PB, somatic embryos at the mid-cotyledonary stage are cocultivated with Agrobacterium containing the desired gene of interest along with a, non-PB, enhanced green fluorescent protein/neomycin phosphotransferase II (egfp/nptII) fusion gene. Modified cultures are grown on proliferation and development medium to produce uniformly modified somatic embryos via secondary embryogenesis. Modified embryos identified on the basis of green fluorescence and kanamycin resistance are transferred to germination medium for plant development. The resulting plants are considered to prototype examples of the PB approach, since they contain egfp/nptII, a non-grapevine-derived fusion gene. Uniform green fluorescent protein (GFP) fluorescence can be observed in all tissues of regenerated plants.

Optimization of Somatic Embryogenesis in Ferula assa-foetida L. (a Rare Medicinal Plant)

Experiments was carried out to examine the effect of abscisic acid (ABA), source of carbon and temperature on secondary somatic embryogenesis and somatic embryo germination of Ferula assafoetida L . Cotyledonary somatic embryos were cultured on MS (Murashig and Skoog, 1962) medium supplemented with 0, 1.0, 1.5 or 2.0 mg/l ABA and on MS medium with 3% mannitol, sorbitol or sources. The highest number of secondary somatic embryos (35.94) was observed on MS medium devoid of ABA at 25°C, but the addition of abscisic acid, sorbitol or manitol to the MS basal medium significantly reduced the formation of secondary somatic embryos at 5°C. The concentration of abscisic acid had a significant effect on the rate of secondary somatic embryogenesis. Among the carbon source studied, mannitol significantly suppressed the induction of secondary somatic embryos. Abscisic acid had a positive effect on the percentage of somatic embryo germination. Higher germination was observed with1.0 or 1.5 mg/l ABA, but it was not significantly affected by sorbitol or manitol. Abscisic acid increased germination at 15°C and 25°C while sorbitol and manitol enhanced it at 5°C.

Direct somatic embryogenesis with high frequency plantlet regeneration and successive cormlet production in saffron (Crocus sativus L.)

Saffron is a slowly propagating geophyte with fungal infestation of corms under field conditions. Moreover, genetic improvement through breeding is not possible due to male sterility. Therefore, tissue culture methods offer a great potential for mass propagation, and somatic embryogenesis is one of the efficient methods. In the present study, direct somatic embryogenesis from leaf base was obtained in thidiazuron (TDZ, 2.5μM) and picloram (2.0μM) supplemented Murashige and Skoog medium (Murashige and Skoog, 1962). Significantly higher secondary embryogenesis was observed in embryo proliferation medium (EPM; MS supplemented with TDZ 2.5μM and picloram 1.0μM). The origin and different developmental stages of somatic embryos were ascertained through histological and scanning electron microscopic studies. Stomata were also observed in some of the somatic embryos. An alluring observation was retention of embryogenic potential beyond 3years of culture. Percent germination of secondary embryos in MS medium was 60.48 when matured in ½ EPM. Freshly initiated somatic embryos also germinated to form plantlets without undergoing secondary embryogenesis in MS medium containing TDZ (2.5μM) and picloram (1.0 and 2.0μM). Somatic embryo derived shoots were multiplied in 6-benzylaminopurine (BAP; 26.64μM) and α-naphthaleneacetic acid (NAA; 1.0μM). Growth performance of cormlets obtained from these shoots was evaluated under green house conditions. Somatic embryogenesis holds tremendous importance for mass propagation of saffron with formation of ultimate propagating material i.e., cormlets.