In vitro regeneration of Camellia reticulata by somatic embryogenesis

Camellia reticulata L. plantlets were regenerated by direct and indirect somatic embryogenesis from immature zygotic embryos. Initial explants (cotyledon sections and embryonic axes) produced somatic embryos without intermediate callus tissue when grown on Murashige and Skoog’s basal medium with 30 gl^-1 sucrose and no growth regulators; the somatic embryos completed their development in 4-6 weeks in the same medium. Embryogénie competence was increased by 0.5 and 1 mg l^-1 IBA. Histological observation showed the embryos to originate from epidermal and subepidermal cells of the cotyledon and hypocotyl explants. Secondary somatic embryos developed directly from the cotyledons and hypocotyl region of primary somatic embryos by a process that was morphologically very similar to that occurring on zygotic explants. Direct repetitive embryogenesis was maintained by this system. Up to 40% germination occurred when mature somatic embryos were isolated and incubated in medium supplemented with 1 mgl^-1 GA₃ + 1 mgl^-1 IAA. Indirect somatic embryogenesis was induced in callus differentiated on cotyledon explants after three months’ culture in media containing IBA or NAA and/or BAP, embryogenic capacity being retained by callus subcultured on 0.5 mg l^-1 IBA + 1 mg l^-1 BAP.     

Evaluation of the efficiency of somatic embryogenesis in guava (Psidium guajava L.)

Summary

An embryogenic protocol for plant regeneration of guava (Psidium guajava L.) was established using 10-week post-anthesis, zygotic embryo explants. Somatic embryogenesis was induced on Murashige and Skoog medium (MS) containing 3% (w/v) sucrose, 0.8% (w/v) agar and various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) by continuous treatment of the zygotic embryo explants. Somatic embryos appeared as globular structures at the end of the third week from culture initiation, and heart-shaped, cotyledonary-stage, and torpedo-stage embryos appeared within the next few weeks. The development of somatic embryos was asynchronous and showed five-to-seven discernible stages. Depending upon the response of the somatic embryos during their maturation, germination, acclimatisation, and encapsulation, they were grouped into one of three categories. The preferred type of somatic embryos (≥ 1.5 mm) were called the “elongated torpedo” (ET) category. The slightly less-preferred type of stomatic embryos (from 1.0 – 1.5 mm) were termed the “short torpedo” (ST) category. The least preferred types of somatic embryos, at the cotyledonary, heart-shaped, and/or globular stages of development (< 1.0 mm), were grouped into a third category designated “CHG”. The suitability and efficacy of various growth regulators and other treatments were assessed based on six different embryogenic parameters: (i) the frequency of embryogenesis; (ii) the intensity of embryogenesis, defined as the average number of somatic embryos produced per culture (“ANEPC”); (iii) the frequency of ET somatic embryos; (iv) the frequency of ST somatic embryos; (v) the frequency of CHG somatic embryos; and (vi) the overall efficiency of embryogenesis, defined as the potential of a treament to produce somatic embryos at the ET or ST stages, or at both stages of development, that could be converted into plantlets. In the present report, we found that 0.01 mg l^–1 2,4-D gave the maximum frequency and intensity of embryogenesis. But the highest frequencies of ET and ST somatic embryos were produced on MS medium containing 3% (w/v) sucrose and 0.001 mg l^–1 2,4-D, while CHG embryos were produced at the highest frequency on the same medium, but containing 0.5 mg l^–1 2,4-D. It was difficult to calculate the most effective concentration of 2,4-D for somatic embryogenesis based on parameters (i) – (v) above. Hence, quantitative estimations of the efficiency of embryogenesis (sixth parameter) were imperative in order to analyse the potential of the different treatments. The highest efficiency of somatic embryogenesis was achieved by continuous treatment of 10-week post-anthesis, zygotic embryo explants with 0.01 mg l^–1 2,4-D on full-strength MS agar medium containing 3% (w/v) sucrose. These somatic embryos matured normally on the same medium, and germinated well both on half-strength solid and in half-strength liquid MS medium containing 3% (w/v) sucrose. They grew in full-strength liquid MS medium with 3% (w/v) sucrose and showed maximum survival upon transfer to soil and hardening. Evaluations of the efficiency of somatic embryogenesis in guava, based on the six parameters defined above, have also helped us to understand and evaluate processes for high efficiency micropropagation in other species.     

Influence of high concentrations of cytokinins on the production of somatic embryos by germinating seeds of tomato,aubergine and pepper

Summary

Somatic embryos of tomato, aubergine and pepper were initiated from intact seedlings when seeds were cultured on medium containing 6-benzylaminopurine (BAP) or thidiazuron (TDZ). The percentage of explants producing somatic embryos was highest for aubergine on media containing low concentrations of BAP, i.e. 0–10 mg l^–1, for tomato at 15–20 mg l^–1 and for pepper at 40–80 mg l^–1. Aubergine and tomato produced fewer somatic embryos per responsive seedling when cultured on medium containing TDZ, and pepper did not produce any somatic embryos on media containing 0–20 mg l^–1 1 TDZ. Morphogenesis of the seedlings producing somatic embryos was similar for all the genotypes, i.e. the seedlings were dwarf, only the cotyledons expanded, development of the apical meristem and the root were suppressed and a ring-like crown of nodular tissue developed at the base of the hypocotyl from which somatic embryos were initiated. Co-cultivation of tomato and aubergine seeds with seeds of pepper in media containing 0, 5, 10, 15 and 20 mg l^–1 BAP inhibited somatic embryogenesis in tomato and aubergine instead of assisting somatic embryogenesis in pepper. This is discussed in relation to the recent findings for the induction of somatic embyrogenesis in peanut (Arachis hypogea L.) and the role of BAP and TDZ.     

Recurrent somatic embryogenesis and plant regeneration in Coriandrum sativum L.

An efficient method of repetitive somatic embryogenesis and plant regeneration was established in Coriandrum sativum L. Embryogenic callus was induced from cotyledon and hypocotyl segments on Murashige and Skoog (MS) medium with 4.52 μM 2,4-dichlorophenoxy acetic acid (2,4-D), upon subculturing on medium having same level of 2,4-D at an interval of 3 weeks developed somatic embryos, which progressed to cotyledonary stage through early developmental stages of somatic embryogenesis. The transfer of somatic embryos at an early cotyledonary and cotyledonary stage in clumps in succession to fresh 4.52 μM 2,4-D supplemented medium developed embryos in a cyclic manner. Upon transferal to embryogenic clumps (cotyledonary embryos) to modified MS medium (4 g l⁻¹ KNO₃, 0.29 g l⁻¹ NH₄NO₃, 3 mg l⁻¹ thiamine HCl, 0.5 mg l⁻¹ pyridoxine HCl, and 5 mg l⁻¹ nicotinic acid), the embryos irrespective of the cycles underwent maturation and germination. Germinating embryos transferred to half-strength MS medium favored healthy growth of plantlets. The system of recurrent somatic embryogenesis in coriander offers a system for genes transfer and also scale-up production of modified plants.     

Direct somatic embryogenesis from explants obtained from in vitro germinated embryonic axes of Camellia sinensis (L.) O. Kuntze

Summary

Studies on direct somatic embryogenesis in several types of explant from in vitro plantlets of tea cultivar TRI 2025 were undertaken to select those most suitable to induce cotyledonary-type somatic embryos. Mature zygotic embryonic axes were surface-sterilised and cultured on MS medium without growth regulators containing 0.6% (w/v) agar. Results showed that 65% of embryonic axes that converted into plantlets at the fifth week of culture had succulent leaves. Several types of explant (normal leaves, large and small succulent leaves, hypocotyl segments and root tips) were isolated from in vitro plantlets at the fifth week and cultured on half-strength MS medium containing 2 mg l^–1 6-benzylaminopurine (BAP) and 0.2 mg l^–1 naphthalene acetic acid (NAA). Morphological and histological observations on somatic embryogenesis were made. The results indicated that somatic embryos were produced at high frequency (25 – 50%) directly from the surface of hypocotyl segments (HS) and large succulent leaves (LSL) after 6 weeks of culture. Efficient somatic embryogenesis was induced in small succulent leaves (SSL) after 16 weeks. Most somatic embryos originated directly from the cortical tissues of HS or the upper epidermal layers of SSL or LSL. HS and SSL from in vitro plantlets gave the highest production of typical, firm somatic embryos for use in tea improvement programmes and for in vitro conservation of tea germplasm.     

Direct somatic embryogenesis in Epipactis veratrifolia,a temperate terrestrial orchid

Most temperate terrestrial orchids are endangered species. Attempts to produce plantlets from plants of the genus Epipactis by asexual methods have totally failed. This study was conducted using somatic embryogenesis as a rapid vegetative propagation technique for conservation of E. veratrifolia. For these purposes, effects of different types of plant growth regulators (PGRs), different types of explants, light and dark conditions, and the effect of gibberellic acid (GA₃) and Paclobutrazol (PBZ) were investigated on somatic embryogenesis induction. Abscisic acid (ABA) pre-treatment effectiveness on inducting somatic embryogenesis and increasing the number of embryos per explant were investigated. Subsequently, 16 media were tested to find the best medium for plant regeneration and shoot and root proliferation. BA (3 mg L^?1) resulted in a better response than the other PGRs by supporting the development of 17 embryos per protocorm. PBZ, which resulted in 11 embryos per explant, was better than GA₃. FAST medium supplemented with organic substances was recognized as the best medium for plant regeneration and shoot and root proliferation. ABA pre-treatment had positive effect on somatic embryogenesis initiation. This study, for the first time, succeeded in finding a rapid and suitable protocol for propagation and genetic resource conservation of E. veratrifolia.     

Improvements in somatic embryogenesis protocol in Feijoa (Acca sellowiana (Berg) Burret): Induction,conversion and synthetic seeds

Pineapple guava (Acca sellowiana) syn. Feijoa sellowiana, a Brazilian indigenous Myrtaceae is under domestication in South Brazil. Previous works showed that this species is responsive to somatic embryogenesis and recalcitrant to conventional methods of clonal propagation. In the present work it was evaluated the role of components of culture medium in the induction and development of somatic embryos. The technology of synthetic seeds was also evaluated. Zygotic embryos were inoculated in LPm medium supplemented with 8 mM glutamic acid and 8 mM l-glutamine, 2,4-dichlophenoxiacetic acid (20 μM) and myo-inositol. For conversion of somatic embryos and synthetic seeds it was tested the effect of 6-benzylaminopurine and gibberellic acid combined or not with activated charcoal. The highest values for embryogenetic induction (100%) and number of somatic embryos/explant (113) were observed in the LPm medium supplemented with Glu (8 mM), and 2,4-D. The culture medium supplemented with BA (0.5 μM) and GA₃ (1 μM) and activated charcoal (1.5 g L⁻¹) enhanced the conversion of somatic embryos to plantlets. Pre-germinated somatic embryos encapsulated in sodium alginate with BA (0.5 μM) and GA₃ (1 μM) developed radicles. The use of synthetic seed was a requisite for the survival of plantlets.     

Direct somatic embryogenesis and plant regeneration from leaf cultures of ornamental species of Dianthus

Protocol for direct somatic embryogenesis from leaf explants of economically important species of Dianthus, viz. D. caryophyllus, D. barbatus and D. chinensis has been developed. Murashige and Skoog’s (MS) liquid medium supplemented with 2,4-D (1 mg/l) was used for direct induction of somatic embryogenesis without an intervening callus phase. Initially globular structures were observed after 21 days of culture of leaf explants in liquid medium. Development of embryos to heart and torpedo stages was achieved in the liquid medium incorporated with polyethylene glycol (PEG 6000) at a concentration of 2.5%. Embryo maturation was further promoted by addition of casein hydrolysate (CH) (200 mg/l) in MS liquid medium. Embryos germinated to form plantlets on solid MS medium supplemented with GA₃ (1 mg/l). Regenerated plants with well-developed root and shoot systems were successfully transferred to field conditions.     

Assessing the influence of subcultures and liquid medium during somatic embryogenesis and plant regeneration in oil palm (Elaeis guineensis Jacq.)

The current analysis describes an improved protocol for somatic embryogenesis and plant regeneration in oil palm (Elaeis guineensis) through liquid medium, and assesses the influence of successive subcultures during induction of calluses in three Brazilian oil palm varieties. Calluses were induced in a Murashige and Skoog (MS) medium with 450 Picloram, 0.5 g L^?1 glutamine, 2.5 g L^?1 activated charcoal, 30 g L^?1 sucrose, and solidified with 2.5 g L^?1 Phytagel. In a first experiment, the effect of continued subculture of explants every 30 days to fresh culture medium was determined. During a second experiment, part of the embryogenic calluses obtained were transferred to a liquid medium under agitation, consisting of MS with 5 µM picloram or 2,4-dichlorophenoxyacetic acid (2,4-D). After 210 days, the calluses were transferred to semi-solid media for differentiating somatic embryos. It was observed that continued subculture of explants monthly was a determinant in stimulating and improving the formation of embryogenic calluses. Embryogenic calluses in liquid medium with 2,4-D significantly improved the percentage of differentiated somatic embryos (up to 80.2%), with the largest amount of torpedo embryos (8.3 per callus). Regenerated plants with roots were individualised and transferred to a greenhouse, with close to 95% survival.     

Effect of ABA and sucrose on germination of encapsulated somatic embryos of guava (Psidium guajava L.)

Present study demonstrates the effect of sucrose and ABA on germination of encapsulated somatic embryos of guava (Psidium guajava L.). Sucrose and ABA at different concentrations were also evaluated for their effects on maturation and germination of somatic embryos. Mature somatic embryos developed on MS medium containing high concentration of sucrose (10%) or ABA (1.0 mg l⁻¹) showed inhibition in germination if they continued to be in same medium for 4 weeks. With increasing concentrations of sucrose (3–9%) or ABA (0.01–1.0 mg l⁻¹) in medium, percent germination of encapsulated somatic embryos decreased significantly. Encapsulated somatic embryos after storage on MS medium supplemented with 9% sucrose or 1 mg l⁻¹ ABA for different duration (0–60 days) germinated when they were transferred to medium containing 3% sucrose. About 20.8% and 37.5% encapsulated somatic embryos germinated after storage on ABA (1 mg l⁻¹) or sucrose (9%) for 60 days, respectively. Temporarily suppression in germination of encapsulated somatic embryos by high concentration of sucrose or ABA may be important for short-term conservation of elite genotype of guava.     

Recurrent somatic embryogenesis and plant regeneration in Angelica glauca Edgew., a critically endangered medicinal plant of the Western Himalaya

Summary

Secondary somatic embryogenesis and plant regeneration from seedling explants of Angelica glauca, an endangered medicinal plant of the Himalaya, is reported for the first time. Callus was obtained from all the explants tested in the present study (i.e., epicotyls, hypocotyls, and cotyledonary nodes). The highest frequency of callus formation (95.8%) was observed using epicotyl explants on 4.0 µM 2,4-dichlorophenoxyacetic acid (2,4-D), whereas 70.8% of hypocotyl explants, and 58.3% of cotyledonary nodes produced callus. One-hundred percent embryogenic callus was induced from epicotyl explants in 2.0 µM 6-benzyladenine (BA) and 2.0 µM μnaphthaleneacetic acid (NAA), together with the maximum number of somatic embryos (34.2 embryos per explant). Cotyledonary nodes did not produce somatic embryos. Histological studies confirmed the induction of somatic embryogenesis. Somatic embryos germinated into plantlets upon transfer to half-strength Murashige and Skoog (MS) medium without added plant growth regulators. We observed 85% survival of these plantlets under field conditions. The development of secondary embryos was also observed when primary embryos were sub-cultured on full-strength MS medium containing 2.0 µM NAA plus 2.0 µM BA. This system of recurrent somatic embryogenesis provides a route for gene transfer and also for the large-scale production of this critically endangered medicinal plant.     

Somatic embryogenesis and plantlet regeneration from encapsulated embryos of Selinum tenuifolium

Summary

This paper reports, for the first time, somatic embryogenesis and synthetic seed production in Selinum tenuifolium Wall. Mature leaf explants inoculated in Murishige and Skoog (MS) medium supplemented with 3 µM 2,4-dichlorophenoxyacetic acid (2,4-D), containing 3% (w/v) sucrose and 0.7% (w/v) agar, induced 67% callus. Maximum production of globular structures, their differentiation into embryos and germination, occurred with a combination of 2 µM benzyladenine (BA) and 2 µM indole-3-butyric acid (IBA). To protect somatic embryos and produce synthetic seeds, gel capsules were standardised using a combination of sodium alginate and calcium nitrate concentrations. Gel capsules were most effective when formed with a combination of 3% (w/v) sodium alginate and 100 mM calcium nitrate for 30 min. The addition of MS medium to alginate capsules with 3% (w/v) sodium alginate, 3% (w/v) sucrose, 2 µM BA and 2 µM IBA significantly improved their germination rate to 77.8%, as well as their resulting shoot length (5.6 cm) and root length (7.2 cm), compared to controls (57.8%). Most plantlets (66%) survived under nursery condition. Storage at 4°C for different periods (10 d or 20 d) significantly (P < 0.05) reduced the percentage survival and germination of somatic embryos and artificial seeds compared to controls or 5 d storage.     

Optimization of somatic embryogenesis in suspension cultures of horsegram [Macrotyloma uniflorum (Lam.) Verdc.]—A hardy grain legume

Cell suspension cultures were established from immature cotyledon derived calli from drought tolerant legume horsegram [Macrotyloma uniflorum (Lam.) Verdc.]. Embryogenic callus could be originated from cut slices of the immature cotyledons on MS solid medium [Murashige, T. Skoog, K., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473–497] augmented with 1.0 μM zeatin and 4.5 μM NAA. Numerous somatic embryoids (26.4%) appeared on MS liquid basal nutrient medium with 5.6 μM NAA and with absence of zeatin during 3 weeks culture. Sustained cell division resulted in the formation of cell aggregates, and then progressed to globular, heart and further if they differentiate properly to torpedo and cotyledonary stages within 5 weeks. Transfer of individual embryos on to a fresh MS basal medium with no plant growth regulators was able to achieve complete maturation. Only a relatively few number of embryos developed into root/shoot when transferred to 0.9 μM GA₃, 15 g/l⁻¹ sucrose and 2.4 g/l⁻¹ gelrite containing medium. Substitution of sucrose associated with the use of l-glutamine gave, in the range of concentrations tested, the strongest enhancement of the embryo growth and development. About 5% of somatic embryos were converted into true-to-type fertile plants.     

Plant regeneration of Carica papaya L. through somatic embryogenesis in response to light quality,gelling agent and phloridzin

Difficulties to develop an easy and reproducible protocol to get healthy and well formed plants from somatic embryos of papaya (Carica papaya L.) had included low germination, callus production at the base of the embryo radicle and the occurrence of hyperhydric plantlets among others, and by consequence unsuccessful transfer to the field. With the aim of improving a propagation method, the effects of light quality, gelling agent and phloridzin concentration on the germination of somatic embryos of hermaphrodite C. papaya L. var. Maradol were studied. Somatic embryos were grown on half strength MS medium, with the addition of Chen vitamins [Chen, M.H., Wang, P.J., Maeda, E., 1987. Somatic embryogenesis and plant regeneration in Carica papaya L. tissue culture derived from root explants. Plant Cell Rep. 6, 348–351], solidified with three distinct gelling agents: Sigma^® Agar–Agar, Difco^® Bacto agar and Phytagel^®; supplemented with phloridzin and exposed to different light qualities: blue (54 μmol m⁻² s⁻¹), red (65 μmol m⁻² s⁻¹), gro-lux (68 μmol m⁻² s⁻¹), red + blue, white (32 μmol m⁻² s⁻¹) and wide spectrum (49 μmol m⁻² s⁻¹) during a period of 4 weeks. Results show that light quality and gelling agent had important effects on germination and plant growth, while 3.0 mg L⁻¹ phloridzin had an important role on germination as well as in root development. Somatic embryos exposed to white light, culture medium solidified with 3.0 mg L⁻¹ phytagel and 3.0 mg L⁻¹ phloridzin showed longer roots. Meanwhile, germination and plant length were promoted on an improved culture medium solidified with 7.5 g L⁻¹ Difco^® Bacto agar, 3.0 mg L⁻¹ phloridzin and exposed to gro-lux lamps. Under these conditions, 70% of somatic embryos germinated and developed normal roots without hyperhydricity. The regenerated plantlets with well developed roots and shoots were successfully transferred to a greenhouse with a survival rate of 95%.     

Histology of somatic embryogenesis in mature tissues of olive (Olea europaea L.)

Summary

This anatomical investigation on olive secondary somatic embryos describes several aspects of embryo development, including proembryoid origin and growth, aspects of tissue differentiation, localization of somatic embryogensis, and starch occurrence. The failure of a number of secondary somatic embryos to develop into perfect structures is to be ascribed to defects in the last growth stages (fused embryos, fused cotyledons) and/or to tissue degeneration processes affecting both imperfect and apparently perfect somatic embryos.     

Direct somatic embryogenesis and adventitious shoot formation from immature axillary buds of Melastoma affine

Summary

Direct somatic embryogenesis and adventitious shoot formation were induced from immature axillary bud explants of Melastoma affine cultured on induction medium containing both naphthaleneacetic acid (NAA) and cytokinins. Among the cytokinins tested, thidiazuron (TDZ) played a greater role in the induction of somatic embryogenesis than 6-benzylaminopurine (BA) and kinetin (KT). Histological studies of paraffin sections showed that the same tissue from immature axillary buds produced different types of explants that developed floral primordia and vegetative bud primordia during the earlier and later flowering stages, respectively. These could result in different developmental pathways. Efficient mass propagation and plant regeneration systems were established for Melastoma affine.     

扁桃杧(Mangifera persiciformis)体胚发生及再生体系建立

以扁桃杧(Mangifera persiciformis Wu&Ming)未成熟珠心组织为外植体,建立体胚发生及再生体系,同时对幼苗进行茎尖染色体计数和胚根组织形态学观察。结果表明,在改良的B5基本培养基+2,4-D1.0mg·L-1+Gln400mg·L-1+6%蔗糖上培养4～5周后可诱导胚性愈伤组织分化。继代培养基与成熟培养基交替培养能有效降低胚性愈伤组织的褐化并保持旺盛的分化能力。培养3～4个月后,大部分体胚均能发育成熟,26.03%的体胚畸形。体胚在改良B5培养基+Gln400mg·L-1+4%蔗糖上的萌发率较低,仅为8.39%。次级体胚以直接体胚发生方式于萌发体胚的下胚轴产生。幼苗的生根不理想,生长极为缓慢;其茎尖染色体数目为2n=2x=40;胚根形态学上端内部维管组织解体,愈伤化,结构松散。     

Improved efficiency of somatic embryogenesis from zygotic embryos in Hyoscyamus niger by seed water-soaking

We describe an efficient procedure to obtain somatic embryos from mature zygotic embryos of Hyoscyamus niger (black henbane). It has several advantages over previous regeneration methods, which are: the use of mature seeds, an average 80% somatic embryogenesis rate and a high (eight-fold higher than the control) plant regeneration frequency. The critical step in this protocol was soaking of the seeds in sterile distilled water for 16 h, prior to excision and culture of zygotic embryos, on MS basal medium supplemented with 2% sucrose, 2 g/l myo-inositol, 0.5 g/l 2-(N-morpholino) ethanesulfonic acid and 1 mg/l α-naphthalene acetic acid (NAA). The regenerated somatic seedlings were fertile and were morphologically uniform. This procedure is simple, rapid and effective for high frequency of plant regeneration via somatic embryogenesis. Moreover, this new method should facilitate the development of strategies to routinely transform recalcitrant plant species, including henbane.     

Somatic embryogenesis of Limonium sinense: a study on the regeneration efficacy via direct and indirect approach followed by Agrobacterium-mediated genetic transformation

An efficient indirect somatic embryogenesis and Agrobacterium-mediated transformation protocol for Limonium sinense has been established, wherein neomycin phosphotransferase II (npt II) and β-glucuronidase (GUS) genes were used as selectable and screenable markers, respectively. The efficiency of plantlet regeneration from transformed tissue was compared between direct embryogenesis from leaf and indirect embryogenesis from callus. Embryogenic callus (EC) was initiated from leaf explants on MS medium supplemented with 6.7 μM 2,4-D and 2.22 μM BA. The somatic embryos were induced, matured, and germinated when ECs were transferred onto MS medium supplemented with 4.44 μM BA and 1.07 μM NAA. Agrobacterium tumefaciens strain LBA 4404 containing the vector pBI121 was used for the transformation. Transient GUS expression frequency was evaluated and putative transgenic plants were successfully grown on culture medium in presence of kanamycin (80–100 mg L^?1). PCR analysis of putative transgenic plants confirmed the presence of GUS and nptII genes. The transformation efficiency obtained through indirect embryogenesis from calluses (4%) was much higher than through direct embryogenesis from leaf explants (0.9%).     

Direct somatic embryogenesis and plant regeneration from leaf explants of Limoniumsinensis (Girard) Kuntze

The potentialities of direct somatic embryogenesis and plant regeneration from leaf explants of Limoniumsinensis var. Golden Diamond invitro were investigated. Young whole leaf and cut leaf explants when cultured on MS basal medium supplemented with each of the growth regulators N⁶-benzyladenine (BA) (0.44–2.2 μM) or thidiazuron (TDZ) (4.54 μM) alone or in combination with a fixed concentration of α-naphthalene acetic acid (NAA) (1.07 μM) produced somatic embryos directly. More than 90% of the leaf explants produced white, globular somatic embryos on BA (2.2 μM) and NAA (1.07 μM) supplemented MS basal medium within 1 week of inoculation. Most of the embryos matured further and converted after 8 weeks of culture on the same medium. Histological observation showed that the somatic embryos originated from single cells of epidermal layer of leaf. Histological evidence of formation of shoot and root poles during conversion of the embryos confirmed that these structures were true somatic embryos. After conversion the plantlets were further placed on MS medium containing 0.44 μM BA and 4.5 μM IBA for better shoot and root growth. About 90% of the plantlets transferred to the mixture of soil:perlite:vermiculite (1:1:1) in small plastic pots acclimatized successfully. Of these 85.5% plants survived after transferring into earthen pots containing a mixture of soil, coarse sand and cattle manure (1:1:1) under greenhouse or shady open condition.