Plant regeneration through somatic embryogenesis on Holostemma ada-kodien,a rare medicinal plant

Plant regeneration through indirect somatic embryogenesis has been established on Holostemma ada-kodien Schult. Type of auxin significantly influenced somatic embryogenesis. Friable callus, developed from leaf, internode and root explants on Murashige and Skoog (MS) medium supplemented with 2,4-D (1.0 mg l^–1), was most effective for the induction of somatic embryos. Subculture of the friable callus developed on 2,4-D (1.0 mg l^–1) onto solid or liquid 1/2 MS medium with 0.1 or 0.5 mg l 2,4-D turned the callus embryogenic. Suspension cultures were superior to static cultures (solid medium) for the induction of somatic embryos. Transfer of embryogenic callus to liquid 1/2 or 1/4 MS medium with lower levels of 2,4-D (0.05–0.1 mg l^–1) induced the highest number of somatic embryos. An average of 40 embryos were obtained from 10 mg callus. Fifty per cent embryos exhibited maturation and conversion upon transfer to 1/10 MS basal solid medium. Plantlets were established in field conditions and 90 per cent survived.     

Regeneration of Acacia mangium through somatic embryogenesis

 Somatic embryogenesis and whole plant regeneration were achieved in callus cultures derived from immature zygotic embryos of Acacia mangium. Embryogenic callus was induced on MS medium containing combinations of TDZ (1–2 mg/l), IAA (0.25–2 mg/l) and a mixture of amino acids. Globular embryos developed on embryogenic callus cultured on the induction medium. Nearly 42% of embryogenic cultures with globular embryos produced torpedo- and cotyledonary-stage embryos by a two-step maturation phase. The first stage occurred on 1/2-strength MS basal medium containing 30 g/l sucrose and 5 mg/l GA₃ followed by the second stage on 1/2-strength MS basal medium containing 50 g/l sucrose. Of the cotyledonary-stage somatic embryos, 11% germinated into seedlings that could be successfully transferred to pots. Light- and scanning electron microscopy showed that the somatic embryos originated from single cells of the embryogenic callus. Further, a single cell layer could be detected beneath the developing somatic embryos that appeared to be a demarcation layer isolating the somatic proembryonic structure from the rest of the maternal callus. A suspensor-like structure connected the globular embryos to the demarcation layer. This is the first successful report of plant regeneration through somatic embryogenesis for this economically important tropical forest species. Received: 20 January 2000 / Revision received: 28 September 2000 / Accepted: 29 September     

Regeneration of pineapple (Ananas comosus L.) plant through somatic embryogenesis

An efficient protocol for a complete plant regeneration by somatic embryogenesis was developed with Smooth Cayenne pineapple (Ananas comosus L.). Previous works showed that this species is responsive to somatic embryogenesis. In the present work the influence of components of culture medium in the induction, development and conversion of somatic embryos was investigate in order to establish a somatic embryogenesis protocol. Nodular callus (83.67%) was initiated from leaf explants of young plants on CIM₃ medium. The highest frequency (37.6%) of embryogenic callus induction was obtained from 4-week-old calluses on EIM₃ medium supplemented with 3.0 mg/l picloram. The highly organized callus induction and the development of somatic embryos were achieved after the transfer of callus clumps onto EIM₃ medium containing 1.0 mg/l BAP + 0.1 mg/l NAA. The frequency of somatic embryo formation was of 39.5?±?2.45 embryos per callus. Up to 97% of the somatic embryos were converted into complete plants within 4 week on MSB medium with 1.0 mg/l BAP + 0.05 mg/l GA₃ + 500 mg/l glutamine. The continuation of the elongation of the shoots occurred on this medium). Shoots obtained from all the above methods were rooted in MSB medium with activated charcoal. Complete plantlets were transferred onto specially made polyethylene bags containing soil mixture and transferred to the greenhouse. Survival rate of the plantlets under ex vitro conditions was 98% and maximum average number of plantlets (80?±?0.6). The well-developed plantlets were transferred to an open field where the plants produced normal fruits.     

Regeneration of pineapple plants via somatic embryogenesis and organogenesis

Summary We have developed efficient methods for plant regeneration, via both embryogenesis and organogenesis, of Smooth Cayenne pineapple, Ananas comosus (L.) Merr. Leaf bases and core (stem) sections of in vitro shoots, produced from culture of crown tip meristem, were used as explants for plant regeneration as follows: (1) Leaf base and core section explants cultured on Murashige and Skoog (MS) medium containing 41 μM 4-amino-3,5,6-trichloropicolinic acid (picloram, P) or thidiazuron (T)/P combinations produced embryogenic tissues. Different types of embryogenic tissues (friable emryogenic tissue, embryogenic cell cluster, and chunky embryogenic tissue) have been developed with varying properties in terms of growth rate and state of development. The embryogenic tissues regenerated shoots upon culture on MS medium containing 13 μM 6-benzylaminopurine (BA) and 1μM α-naphthaleneacetic acid (NAA) followed by culture on MS medium containing 4 μM BA. (2) Crown tip meristems cultured on MS medium containing 13 μM BA followed by leaf explants cultured on MS medium with 27 μM NAA and 1 μM BA produced shoots via direct organogenesis. (3) Explants cultured on MS medium containing 5 μM T and 0.5 μM indole-3-butyric acid (IBA) produced nodular globular structures, which produced shoots upon culture on MS medium containing 1 μM BA and 1 μM gibberellic acid. Shoots obtained from all of the above methods were rooted in half-strength MS medium containing 3 μM NAA and 2.5 μM IBA. Plants were transferred to the greenhouse or shipped to Costa Rica for field trials. Somatic embryo-derived plants exhibited 21 % spininess, and organogenic-derived plants exhibited 5% spininess in the field trials.     

Regeneration and large-scale propagation of Phragmites communis through somatic embryogenesis

A protocol for large-scale propagation of Phragmites communis Trin. by somatic embryogenesis has been established. Plants were regenerated through somatic embryogenesis from stem segments of R5002-12, a salt-tolerant variant line of Phragmites communis Trin. Stem segment explants produced hard white callus on the semi-solid Murashige and Skoog (MS) medium supplemented with 9.05 M 2,4-dichlorophenoxy acetic acid (2,4-D) for 4 weeks. The induction frequency was 36.7%. Then, the callus was transferred to MS medium supplemented with 4.52 M 2,4-D. After 4 weeks in culture, yellow embryogenic callus with some nodular structures was formed. When the embryogenic callus was transferred to differentiation medium (MS supplemented with 0.45 M 2,4-D), differentiation was initiated to form small green islands on the surface of the callus after 2 weeks in culture. Within 4 weeks, a large number of somatic embryos were formed with a frequency of 86.7%. Six weeks later, they developed into strong plantlets. When the plantlets (about 1 cm in length) were cultured on propagation medium (MS supplemented with 13.31 M BA+5.37 M NAA), a great number of regenerated plants were obtained. After the plants were cultured on liquid 1/2 MS medium with 2.69 M NAA added 2.46 M IBA roots developed. The rooted plants were transferred to soil with over 85% survival. Using this methodology, more than 20000 regenerated plants of salt tolerant variant line of Phragmites communis Trin. have been produced.     

Regeneration of Ensete ventricosum through somatic embryogenesis and adventitious buds

In southern and south-western Ethiopia, Ensete ventricosum is grown as an important starchy, staple food crop, supporting the diet of a quarter of the Ethiopian population. Due to difficulty in germinating seeds and the long vegetative period, breeding enset is extremely difficult. Adventitious buds and somatic embryos have been induced from callus derived from corm tissues and cultured on Murashige and Skoog's (MS) basal medium supplemented with benzylaminopurine (BAP) or 6 --dimethylallylamino purine 2iP. Elongation of somatic embryos was achieved on the same medium and rooting was induced on half-strength MS basal medium supplemented with IBA. No phenotypic variation was observed among more than 200 potted regenerants. The possible implications for mutation breeding in this crop are discussed.Abbreviations IAA Indole-acetic acid - IBA Indole-butyric acid - BAP Benzylaminopurine - 2iP 6 --dimethylallylamino purine     

Plantlet regeneration through indirect shoot organogenesis and somatic embryogenesis in Justicia gendarussa Burm. f., a medicinal plant

A protocol for the regeneration of a large number of plantlets via indirect shoot organogenesis and somatic embryogenesis has been developed from the stem and leaf explants of Justicia gendarussa Burm. f. The callus was efficiently induced from the explants using Murashige and Skoog (MS) medium supplemented with α-Naphthalene acetic acid (NAA) + Benzyl amino purine (BAP) (1.0?+?0.1 mg/l). The highest number of plantlets through indirect shoot organogenesis was obtained when the callus was subcultured to MS medium with BAP + NAA (0.1?+?1.0 mg/l). The maximum number of plantlets via somatic embryos was obtained in the medium with BAP + NAA (1.0?+?0.1 mg/l) for stem derived calli and Kinetin (Kn) + NAA (2.0?+?0.1 mg/l) for leaf derived calli. The in vitro developed shoots were rooted well in half strength MS medium supplemented with 0.5 mg/l of Indole-3-acetic acid (IAA). The in vitro regenerated plantlets were hardened using a mixture of sterile sand:soil:manure (1:1:1). The present study is the first report on the regeneration of plants through somatic embryogenesis from stem and leaf derived calli of J. gendarussa.     

Regeneration of transgenic papaya plants via somatic embryogenesis induced byAgrobacterium rhizogenes

AnAgrobacterium rhizogenes-mediated procedure for transformation of papaya (Carica papaya) was developed. Transgenic plants were obtained from somatic embryos that spontaneously formed at the base of transformed roots, induced from leaf discs infected withA. rhizogenes. Transformation was monitored by autonomous growth of roots and somatic embryos, resistance to kanamycin, β-glucuronidase activity (GUS), and Southern hybridization analysis. Over one-third of the infected leaf explants produced transformed roots with GUS activity, from which 10% spontaneously produced somatic embryos. Histological analysis ofA. rhizogenes-transformed embryos showed that they have an altered symmetry between the shoot apex and the root meristem when compared to somatic embryos induced with hormone treatment from control explants. Transgenic papaya plants containingA. rhizogenes rol genes were more sensitive to auxins, developed wrinkled leaves, and grew slower than nontransformed plants.     

Plant regeneration through direct somatic embryogenesis from immature zygotic embryos of the medicinal plant,Paris polyphylla Sm.

A protocol for induction of direct somatic embryogenesis and subsequent plant regeneration for the medicinally important and endangered plant Paris polyphylla Sm. has been developed for the first time. Immature zygotic embryos (IZEs) were cultured on different media namely Gamborg (B5), ½ B5, Murashige and Skoog (MS), ½ MS, Chu et al. (N6), ½ N6, Schenk and Hildebrandt (SH) and ½ SH. Highest frequency of somatic embryogenesis (32.6 %) and mean number of somatic embryos (SEs) per explant (28.7 ± 1.7) were obtained on ½ MS medium directly without an intermediate callus phase. The frequency of SE induction was significantly increased to 40.7 % when ½ MS medium was solidified with gelrite compared to agar (32.6 %). Secondary somatic embryos (SSEs) appeared on the primary SEs in a repetitive way on plant growth regulator-free ½ MS medium but with a gradual decrease in embryogenic potential during subsequent subcultures. Plasmolyzing pre-treatment of SSEs with 1.0 M mannitol for 12 h effectively maintains its embryogenic capacity. Primary embryos at the elongated dimpled and early cotyledonary stage displayed the highest embryo forming capacity of 26.94 and 27.87, respectively. High frequency of SE germination (94.0 %) occurred on ½ MS medium with 0.5 mg/l gibberellic acid. Highest percentage of seedling to plantlet conversion was observed in the medium supplemented with 0.05 mg/l 6-benzylaminopurine and 0.1 mg/l α-naphthalene acetic acid. Regenerated plants displayed morphological characteristics similar to that of the wild plants. Flow cytometry analysis showed ploidy stability of the regenerated plants.     

Repetitive somatic embryogenesis from leaves of the medicinal plant Petiveria alliacea L.

Leaf segments from in vitro-grown shoot cultures of Petiveria alliacea were incubated on Murashige and Skoog (MS) medium supplemented with different concentrations of zeatin, thidiazuron, 2,4-dichlorophenoxyacetic acid (2,4-D) or picloram (PIC). Direct somatic embryogenesis was induced in response to all tested concentrations of 2,4-D and PIC. Primary somatic embryos displayed highly repetitive embryogenesis, both on the induction medium and in liquid hormone-free MS medium. Plantlets were obtained from these secondary embryos at an estimated frequency of 5 %, after 180 days of culture on half-strength MS medium gelled with 0.2 % Phytagel. Simultaneous development of friable non-embryogenic callus was also observed on media containing PIC or 2,4-D at different concentrations. Cell suspension cultures initiated from these callus tissues did not show an increase in biomass. The embryogenic portions formed at the surface of the explants in response to 20.0 μM PIC were inoculated in hormone-free full-or half-strength liquid MS medium (MS0) and showed high rates of secondary embryogenesis, resulting in the production of a mean of 35 embryos for each embryo inoculated at the culture initiation. Embryos that started the conversion process in the liquid MS0 medium originated whole plants at a frequency of 100 % when transferred to MS0 medium solidified with 0.7 % agar. Acclimatization was achieved in 90 % of the converted plantlets, with the production of phenotypically normal plants. This system is potentially useful for the micropropagation of this species, as well as for the production of substances with pharmacological interest, such as dibenzyl trisulfide.     

In vitro plant regeneration through somatic embryogenesis in Anaphyllum wightii Schott

In Vitro Cellular & Developmental Biology - Plant - Anaphyllum wightii Schott. is an ethnomedicinally significant plant endemic to the southern region of Western Ghats. The present study aimed...     

In vitro organogenesis and plant regeneration of Thymus serpyllum L.: an important aromatic medicinal plant

An efficient in vitro propagation system has been developed for the rapid micropropagation of Thymus serpyllum L. (Banajwain), an aromatic medicinal herb from nodal explant on MS medium. Phenolic leaching and high rate of contamination was the most significant problem in establishing in vitro culture of Thymus serpyllum which was overcome by preparing explants in an antioxidant ascorbic acid (1000 ppm) at 6°C for 45 min and addition of the same antioxidant (50 mgl⁻¹) to the MS medium. The frequency of shoot production was influenced by different cytokinins (Kn, BAP, and Kn + BAP) and 95.56% shoot induction was observed when MS medium was supplemented with 1.0 + 2.0 mgl⁻¹ (Kn + BAP). The maximum average number of shoots 16.93 ± 2.15 and average length (3.98 ± 0.55) was recorded when MS medium have 0.5 + 2.0 mgl⁻¹ (Kn + BAP). The in vitro regenerated microshoots were rooted on MS and half strength MS medium and there was significant difference in root induction on both media under the influence of auxins (IAA, IBA, and NAA). The maximum average number (11.67 ± 3.03) and average root length (3.88 ± 0.71) was reported in half MS medium having 1.0 mgl⁻¹ IBA. The complete regenerated plantlets were acclimatized under growth chamber before transferring to the earthen pots and showed 90% survival.

Graphical abstract

     

Induction of somatic embryogenesis in woody plants

Somatic embryogenesis, the in vitro developmental program by which somatic cells are reprogrammed to undergo cellular and molecular changes that make them competent to produce somatic embryos, has been achieved with many woody plants. The program involves the stages of competence acquisition, induction and expression of the morphogenic pathway by the cultured cells and tissues. The ability to express the program in cultured cells/tissues is regulated by many factors, including genotype, explant type and age and culture conditions. In many woody plants, somatic embryogenesis was achieved with mature, immature explants or both. Juvenile tissues as immature and mature zygotic embryos are regarded best explants to establish embryogenic cultures in woody plants and potential to obtain the cultures decline with increasing maturity of the explant.     

Regeneration and large-scale propagation of bamboo (Dendrocalamus strictus Nees) through somatic embryogenesis

A complete protocol for large-scale propagation of Dendrocalamus strictus Nees by somatic embryogenesis has been developed. Seeds cultured on agar-solidified Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D; 3×10^–5 m) produced embryogenic callus from proliferation of the embryo. Somatic embryos formed in vitro multiplied rapidly (two- to five fold every 5 weeks) on semi-solid MS medium containing 2,4-D (1×10^–5 m), kinetin (Kn) (5×10^–6 m), 1-indolebutyric acid (IBA) (2×10^–6 m) and soluble polyvinylpyrrolidone (PVP) (250 mg l^–1), or MS with 2,4-D (1×10^–5 m), 6-benzylaminopurine (BAP) (1×10^–5 m), and soluble PVP (250 mg l^–1). Upon transfer to MS containing 1-naphthaleneacetic acid (NAA) (5×10^–6 m), Kn (5×10^–6 m) and soluble PVP (250 mg l^–1), the dark-green embryos developed into healthy plantlets. Unrooted shoots, if any, obtained on the multiplication media were rooted on MS major salts reduced to half strength supplemented with NAA (3×10^–6 m) and IBA (2.5×10^–6 m). The rooted plants were successfully transferred to soil in polythene bags with over 80% survival. Using this methodology, more than 100,000 plants have been produced. Received: 16 April 1998 / Revision received: 25 September 1998 / Accepted: 10 October 1998     

Characteristics of Bupleurum falcatum plants propagated through somatic embryogenesis of callus cultures

Various characteristics including the saponin content in the root of Bupleurum falcatum plants propagated in vitro through somatic embryogenesis of callus cultures were compared with those of the plants propagated by seeds. The asexually propagated plants had an aerial part of more uniform characteristics than those of sexually propagated ones. However, both the mean and variance of root weight of the former were significantly larger than those of the latter. As for the saponin content of the root on a dry weight basis, there was little difference between the two groups. The amounts of saikosaponins c and d in a root were significantly larger in the asexually propagated plants than in the sexually propagated ones.     

Regeneration of soapnut tree through somatic embryogenesis and assessment of genetic fidelity through ISSR and RAPD markers

Somatic embryogenic system was developed in Sapindus mukorossi Gaertn. using rachis as explants from a mature tree. Explants showed callus initiation on Murashige and Skoog medium supplemented with TDZ (1-Phenyl-3-(1, 2, 3-thiadiazol-5-yl) urea), zeatin or 6-benzylaminopurine. Induction of somatic embryogenesis was achieved on both MS basal medium and MS medium supplemented with 8.88 µM 6-benzylaminopurine. Hundred percent embryogenesis was observed on MS medium supplemented with 8.88 µM 6-benzylaminopurine with maximum intensity of embryogenesis (51.92 ± 0.40 a). Maximum maturation of somatic embryos (92.86 ± 0.34 a) was observed on induction medium supplemented with 0.0378 µM abscisic and treated for 21 days. Germination of somatic embryos was maximum (77.33 ± 0.58 a) on MS medium supplemented with 8.88 µM 6-benzylaminopurine. In vitro raised plantlets were hardened, acclimatized and transferred to the field. Survival frequency of plantlets was 80 % in field conditions. The genetic fidelity of in vitro regenerated plants was also evaluated and compared with mother plant using random amplified polymorphic DNA and inter simple sequence repeat. Both markers showed similarity in molecular profile of mother plant and in vitro regenerated plants.     

植物体细胞胚胎发生的调控网络

植物体细胞胚胎发生是一个极其复杂而有序的过程,受到多种内外因素的影响与调控。其中基因的表达与调控是影响体细胞胚胎发生最重要和最根本的因素。这些基因包括PLANT GROWTH ACTIVATOR系列基因、LEAFYCOTYLEDON家族基因、BABY BOOM基因、SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE基因和PICKLE基因等,它们相互作用构成了一个复杂的调控网络。以下结合作者对PLANT GROWTH ACTIVATOR 37等基因的研究,对这一调控网络进行了介绍,并探讨了未来体细胞胚胎发生的研究方向。     

Secondary somatic embryogenesis and plant regeneration in cassava

Somatic embryos isolated from mature seed-derived cotyledon cultures of cassava (Mannihot esculenta Crantz) underwent direct secondary somatic embryogenesis or plant development under appropriate incubation conditions. Isolated somatic embryos were subjected to a two-stage culture procedure similar to that which induced their development on cotyledon explants. This involved incubation for 24–30 days on Murashige and Skoog basal medium supplemented with 2–8 mgl^-1 2,4-dichlorophenoxyacetic acid (2,4-D) (Stage I medium) before transfer to medium supplemented with 0.01 mgl^-1 2,4-D and 0.1 mgl^-1 6-benzylamino purine (BAP) (Stage II medium). Under these conditions, secondary somatic embryos developed directly from the cotyledons and shoot-tip region of primary somatic embryos by a developmental process morphologically very similar to that occurring on zygotic cotyledon explants. Apical shoot extension and adventitious root formation occurred when somatic embryos were isolated from parental cultures and incubated on Stage II medium. Somatic embryo-derived plants growing in greenhouse conditions appeared morphologically normal when compared with non-regenerated plants.     

Regeneration by somatic embryogenesis of triploid plants from endosperm of walnut,Juglans regia L. cv Manregian

Plants were regenerated by somatic embryogenesis from endosperm tissue of open-pollinated seeds of Juglans regia L. cv Manregian. These plants were obtained by growing endosperm tissue on media similar to those used for plant regeneration from walnut cotyledons (Tulecke and McGranahan 1985). The plants appear morphologically uniform and have a triploid chromosome number of 3n=48. Nine plants have been grown to a young sapling stage in soil. This embryogenic line from endosperm has been maintained in culture for two years by the process of repetitive somatic embryogenesis.     

Regeneration of coconut (Cocos nucifera L.) from plumule explants through somatic embryogenesis

A protocol was developed for coconut regeneration using plumules from mature zygotic embryos as explants, and media with the synthetic growth regulators 2,4-dichlorophenoxyacetic acid and 6-benzylaminopurine. Evidence for the regeneration process from these tissues occurring through somatic embryogenesis is presented. The somatic embryos were capable of germination, subsequent development into plantlets and successful transfer to the nursery. The yields were larger, nearly twofold for calli and over tenfold for calli bearing somatic embryos, than those previously reported with inflorescence explants. The present protocol thus represents an improvement in time and yield over previous protocols. Even though plumule explants are not the ideal tissue source due to possible genetic heterogeneity, the improvements made here may be applicable to tissues from mature plants. In addition, micropropagation of coconut using plumules is potentially useful when they are obtained from fruit produced from selected parents of outstanding performance, such as those resistant to diseases. Received: 19 March 1997 /Revision received: 11 September 1997 / Accepted: 4 October 1997