Somatic embryogenesis and regeneration of Cenchrus ciliaris genotypes from immature inflorescence explants

An efficient, highly reproducible system for plant regeneration via somatic embryogenesis was developed for Cenchrus ciliaris genotypes IG-3108 and IG-74. Explants such as seeds, shoot tip segments and immature inflorescences were cultured on Murashige and Skoog (MS) medium supplemented with 2.0–5.0 mg dm^?3 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg dm^?3 N⁶-benzyladenine (BA) for induction of callus. Callus could be successfully induced from all the three explants of both the genotypes. But the high frequency of embryogenic callus could be induced only from immature inflorescence explants. Somatic embryos were formed from nodular, hard and compact embryogenic calli when 2,4-D concentration was gradually reduced and BA concentration increased. Histological studies of somatic embryos indicated the presence of shoot apical meristem with leaf primordia. Ultrastructural details of globular and scutellar somatic embryos further validated successful induction and progression of somatic embryogenesis. Shoots were differentiated upon germination of somatic embryos on MS medium containing 2,4-D (0.25 mg dm^?3) and BA or kinetin (1–5 mg dm^?3). Roots were induced on ½ MS medium containing charcoal (0.8 %), and the regenerated plants transferred to pots and established in the soil showed normal growth and fertility.     

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.     

Somatic embryogenesis and plant regeneration of Gladiolus (Gladiolus hort.)

Summary Friable embryogenic callus and somatic embryos of 4 Gladiolus cultivars were obtained on Murashige and Skoog (MS) medium with various concentration of auxins from the following explants: corm slices, young leaf bases and whole, intact plantlets. Somatic embryos transferred on MS hormone-free medium regenerated into plantlets. All plantlets obtained through embryogenesis did not differ phenotypically from the parental clones. The embryogenic friable callus has been maintained for over 2 years in culture and has retained a very high regeneration capacity.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - KIN kinetin - NAA naphthaleneacetic acid - MS Murashige and Skoog Medium (1962) - E embryogenic callus - NE non-embryogenic callus     

Friable embryogenic callus and somatic embryo formation from cotyledon explants of African marigold (Tagetes erecta L.)

Embryogenic callus and somatic embryos were induced from cotyledonary explants of African marigold (Tagetes erecta L.). Cotyledons were first cultured on MS medium supplemented with 2.0 mg l^–1 2,4-D and 0.2 mg l^–1 kinetin. After 5 weeks, calli were transferred to MS medium supplemented with 0.02 mg l^–1 thidiazuron where compact embryogenic callus developed. Friable embryogenic callus developed when the compact embryogenic callus was transferred to medium containing 2,4-D and subcultured every 2 weeks. Friable embryogenic callus has been maintained for more than 2 years without losing the capacity to generate embryos. Embryo development was obtained when friable embryogenic callus was transferred to MS medium supplemented with 3 mg l^–1 ABA and 60 g l^–1 sucrose. The addition of 10–30 mM l-glutamine improved embryo development. Received: 13 May 1997 / Revision received: 24 February 1998 / Accepted: 28 March 1998     

The effect of auxins, time exposure to auxin and genotypes on somatic embryogenesis from mature embryos of wheat

The effects of different factors on the embryogenesis and plant regeneration from mature embryos of Russian spring and winter genotypes were studied. Embryogenic callus induction was achieved on MS medium supplemented with different concentrations of 2,4-D (2,4-dichlorophenoxyacetic acid), 2,4,5-T (2,4,5-trichlorophenoxyacetic acid) or Dicamba (3,6-dichloro-o-anisic acid). Although all auxins were able to induce callus from explants with high frequency (98–100%), Dicamba was more effective for the induction of embryogenic callus (21.8–38.3%). Maximum embryogenic callus formation and high number of regenerated plants were observed at 12 mg l⁻¹ of Dicamba. The time exposure to Dicamba (7, 14, 21 and 28 days) had a significant effect on efficiency of somatic embryogenesis. When contact of explants with callus induction medium was increased from 7 to 21 days the rate of somatic embryogenesis and number of regenerated plants per embryogenic callus gradually increased from 13.0 to 38.4% and 3.6 to 8.0%, respectively. Supplement of additional auxins (indoleacetic acid (IAA), indolebutyric acid (IBA), and naphthaleneacetic acid (NAA)) to callus induction medium with Dicamba had a positive effect on the rate of embryogenic callus formation, while the average number of regenerated shoots was not affected. The best rate of somatic embryogenesis was observed at the addition of 0.5 mg l⁻¹ IAA with Dicamba (61.0%). The optimum combination of Dicamba and IAA increased the efficiency of somatic embryogenesis and plant regeneration from seven spring and winter wheat genotypes, thought overall morphogenic capacity was still genotype dependent.     

Induction and Proliferation of Callus Derived from Fruits of Different Varieties of Capsicum annuum

Callus was initiated on Murashige and Skoog (MS) medium containing different combinations of growth regulators or different concentrations of vitamins from pericarp of six varieties of Capsicum annuum differing in their capsaicin content. Callus derived from pericarp of low capsaicin containing varieties was snowy white, friable and showed excellent growth. Callus initiation was delayed (10-15 days) in Punjab Lal, which had very high fruit capsaicin content (7.0 mg g^?1 DW). It also showed relatively slow proliferation although callus obtained was white and friable. Several different media were tried to improve the initiation and the proliferation of the callus in this variety. Callus growth improved greatly by doubling the concentration of MS salts. Initiation time was reduced to 4-6 days by adding 10 mg l^?1 NAA and 0.5 mg l^?1 Kin in MS medium. Other combinations of growth regulators or increase in concentration of vitamins or activated charcoal (0.1%) resulted in poor callus growth and callus texture. Of all media tried, MS medium containing 2 mg l^?1 2,4 D and 0.5 mg l^?1 Kin was the best for callus initiation and proliferation.     

Callus Formation and Plant Regeneration from Cultured Embryos of Diploid and Tetraploid Winter Ryes (Secale cereale L)

The ability of immature, mature and endosperm-supported mature embryos of diploid and tetraploid winter ryes (Secale cereale L) was tested to compare the callus induction and plant regeneration. Immature embryos were obtained from field grown rye. Immature embryos were aseptically excised and placed, with the scutellum upwards, on the callus culture medium consisted of Murashige and Skoog (MS) mineral salts supplemented with 2 mg l^?1 2,4-dichlorophenoxyacetic acid (2,4-D). Mature embryos were aseptically excised the imbibed seeds and placed, scutellum up, on MS medium supplement with 2 mg l^?1 2,4-D. Endosperm-supported mature embryos were moved slightly (not set free) in the imbibed mature seeds. The seeds with moved embryos were placed furrow downwards in dishes containing 8 mg l^?1 2,4-D for callus induction. The developed calli and regenerated plant were maintained on hormone free MS medium. Comparison of the responses of the three explants used indicated that endosperm-supported mature embryo was the most useful explant for plant regeneration in both diploid and tetraplold ryes. This is the first report of winter ryes plants having been regenerated from endosperm-supported mature embryos.     

High-frequency somatic embryogenesis from germinated zygotic embryos of <Emphasis Type="Italic">Schisandra chinensis</Emphasis> and evaluation of the effects of medium strength,sucrose, GA<Subscript>3</Subscript>, and BA on somatic embryo development

We developed a new protocol for highly efficient somatic embryogenesis and plantlet conversion of Schisandra chinensis. Friable embryogenic callus was induced from cotyledonary leaves and hypocotyls of germinated zygotic embryos on Murashige and Skoog (MS) agar medium containing 2,4-dichlorophenoxyacetic acid (2,4-D). Preculture of zygotic embryos on 2,4-D-containing medium increased embryogenic callus induction efficiency. The highest embryogenic callus induction frequency of 56.7% was obtained from shoot apical meristem-containing hypocotyl explants from 1-week-old germinated embryos on MS medium containing 4.0 mg l⁻¹ 2,4-D. Embryogenic callus proliferation, somatic embryo (SE) formation, and subsequent plantlet conversion occurred under optimal culture conditions. The effects of MS medium strength, sucrose, gibberellic acid (GA₃), and 6-benzyladenine (BA) on SE formation and plantlet conversion were evaluated. Low MS medium strength (1/4 to 1/2) was necessary for SE formation, and the optimal sucrose concentration was 2.0%. Supplementing medium with GA₃ negatively impacted SE formation and subsequent development. BA significantly increased the number of SEs and the plantlet conversion capacity. One-third-strength MS medium with 1.0% sucrose and 0.5 mg l⁻¹ BA produced the highest number of SEs (309 embryos from 9 mg embryogenic callus) and the highest frequency of plantlet conversion from germinated SEs (52.6%). When transplanted to soil, 90% of the regenerated plants developed into normal plants.     

Comparison of developmental stages of inflorescence for high frequency plant regeneration in Triticum aestivum L. and T. durum Desf.

Summary Whole immature inflorescences at 4 different developmental stages (0.5, 1.0, 1.5, 2.0 cm in size) of different genotypes of Triticum aestivum and T. durum were cultured to see the morphogenetic responses on Murashige and Skoog's (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) (2.5 mg/l). Very young inflorescences 0.5 and 1.0cm long formed embryogenic callus from their entire surface while 1.5 and 2.0 cm long inflorescences formed embryogenic callus from the basal spikelets and rachis only. This embryogenic callus was maintained by regular subcultures on MS medium with 2,4-D (2.5 mg/l) for more than a year. Plantlets were regenerated by transferring the embryogenic callus on hormone-free MS medium. Inflorescences (0.5 and 1.0 cm long) responded best in forming callus as well as plantlets at a very high frequency. Variation in response was observed amongst the genotypes but the qualitative response of formation of embryogenic callus and later regeneration of plantlets was observed from all the genotypes. Immature young inflorescence explants could provide a suitable material for particle gun mediated genetic transformation in wheat.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - MS Murashige and Skoog (1962)     

Somatic embryogenesis in two Gossypium hirsutum genotypes on semi-solid versus liquid proliferation media

A comparison of semi-solid vs. liquid embryo proliferation media was made using two Gossypium hirsutum L. genotypes (Coker 312 and T25) and two callus initiation media. Sections of petioles from mature, flowering plants were cultured on two modified Murashige and Skoog media. Medium 1 included 4.0 mg l^-1 NAA and 1.0 mg l^-1 kinetin; medium 2 contained 0.1 mg l^-1 2,4-D and 0.1 mg l^-1 kinetin. After six weeks, callus was removed from each explant and divided in half. One callus portion was placed in liquid proliferation medium and the other on semi-solid (0.2% Gelrite) proliferation medium. Composition of proliferation medium was identical to that of initiation medium, except no growth regulators were added. Embryos were counted after eight weeks. The percentage of explants forming callus was influenced by genotype/initiation medium combination. Analysis of variance procedures revealed significant variability for callus initiation media, proliferation media (semi-solid or liquid), and an initiation medium x genotype interaction. Paired t-tests indicated that more embryos were produced in liquid proliferation medium (227.3 embryos/culture) than on semi-solid proliferation medium (134.6 embryos/culture).Abbreviations NAA naphtaleneacetic acid - 2,4-D 2,4-D dichlorophenoxyacetic acid     

<Emphasis Type="Italic">In vitro</Emphasis> selection of NaCl-tolerant callus lines and regeneration of plantlets in a bamboo (<Emphasis Type="Italic">Dendrocalamus strictus</Emphasis> Nees)

Summary Sodium chloride-tolerant plantlets of Dendrocalamus strictus were regenerated successfully from NaCl-tolerant embryogenic callus via somatic embryogenesis. The selection of embryogenic callus tolerant to 100 mM NaCl was made by exposing the callus to increasing (0–200 mM) concentrations of NaCl in Murashige and Skoog medium having 3% (w/v) sucrose, 0.8% (w/v) agar, 3.0 mg l⁻¹ (13.6 μM) 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.5mg l⁻¹ (2.3μM) kinetin (callus initiation medium). The tolerance of the selected embryogenic callus to 100 mM NaCl was stable through three successive transfers on NaCl-free callus initiation medium. The tolerant embryogenic callus had high levels of Na⁺, sugar, free amino acids, and proline but a slight decline was recorded in K⁺ level. The stable 100 mM NaCl-tolerant embryogenic callus differentiated somatic embryos on maintenance medium [MS medium +3% sucrose +0.8% agar +2.0 mg l⁻¹ (9.0 μM) 2,4-D+0.5 mg l⁻¹ (2.3 μM) kinetin] supplemented with different (0–200 mM) concentrations of NaCl. About 39% of mature somatic embryos tolerant to 100 mM NaCl germinated and converted into plantlets in germination medium [half-strength MS+2% sucrose+0.02 mg l⁻¹ (0.1 μM) α-naphthaleneacetic acid +0.1 mg l⁻¹ (0.49 μM) indole-3-butyric acid] containing 100 mM NaCl. Of these plantlets about 31% established well on transplantation into a garden soil and sand (1:1) mixture containing 0.2% (w/w) NaCl.     

Indirect somatic embryogenesis and plant regeneration from leaf and internode explants of Paulownia elongata

Several plant growth regulators BA, TDZ, 2,4- and Kn were tested alone or in combination for their capacity to induce indirect somatic embryogenesis from leaf and internode explants of Paulownia elongata. Calli were produced when leaf explants were cultured on Murashige and Skoog (MS) medium containing 3 % sucrose, 0.4 % phytagel, 4 mg l^-1 TDZ and 0.1 mg l^-1 Kn after 3 weeks and the initiation rate was 54.1%. After subculturing on the same medium, embryos at various developmental stages (globular, heart and torpedo shaped) were transferred for maturation onto MS medium supplemented with 3 % sucrose, 0.4 % phytagel, 0.1 mg l^-1 TDZ, 1 mg l^-1 Kn and 2 mM glutamine. An average of 50.7 somatic embryos were obtained from 100 mg of embryogenic callus after 4 weeks at high frequency (64.7 %). Afterward, mature somatic embryos were isolated and cultured on hormone-free MS medium for germination (80 %) and development into plantlets. Plantlets were transferred to pots with a mixture of peat and perlite in a 3:1 ratio and showed a survival rate of 70–80 %. Plantlets regenerated by this procedure were morphologically identical to the donor material and developed normally in the greenhouse.     

Repetitive somatic embryogenesis in peanut cotyledon cultures by continual exposure to 2,4-d

Somatic embryos from immature cotyledons in peanut (Arachis hypogaea) were initiated on media supplemented with 2,4-dichlorophenoxyacetic acid (2,4-d). Over 90% primary embryogenesis and 41–46% repetitive embryogenesis were obtained 12 weeks after initiation by maintaining embryogenic cultures on medium containing 20 mg 1^-1 2,4-d. Maintenance of cultures on medium with 30 or 40 mg I^-1 2,4-d resulted in lower primary and secondary embryogenesis, and proliferation of nonembryogenic callus. Transfer of embryogenic cultures to a secondary medium with 10 or 20 mg I^-1 2,4-d significantly enhanced secondary embryogenesis compared to basal medium without the growth regulator. The use of Murashige & Skoog versus Finer's media had no significant effect on embryogenesis (85–95%), repetitive embryogenesis (11–37%) or mean embryo number. Secondary embryogenesis was also maintained for over one year by repeated subculture of isolated somatic embryos on medium with 20 mg I^-1 2,4-d.Abbreviations B5 Gamborg et al. medium (Gamborg et al. 1968) - 2,4-d 2,4-dichlorophenoxyacetic acid - FN Finer & Nagasawa medium (Finer & Nagasawa 1968) - MS Murashige & Skoog medium (Murashige & Skoog 1962)     

Long-term optimized embryogenic cultures in durum wheat (Triticum durum Desf.)

Five varieties of durum wheat: Appulo, Ofanto, Latino, Creso, and Castello (Triticum durum Desf.) adapted to the semi-arid mediterranean environment have been tested for their in vitro response. Compact, embryogenic, highly regenerable calli originated from primary callus derived through proliferation of the scutellum of immature embryos explanted in the presence of 2,4 dichlorophenoxyacetic acid. Selective subculture of the white, compact, embryogenic sectors led to the establishment of long-term cultures. Regeneration occurred on hormone-free medium either via germination of somatic embryos, or via multiple-shoot formation probably due to precocious germination of somatic embryos. The three varieties, Ofanto, Creso and Appulo, were the best responding genotypes. Callus fragmentation and two subsequent transfers onto fresh medium at 7-day intervals yielded a frequency of plant regeneration of some 25–40 plantlets per gram fresh weight callus in 21 days on Murashige and Skoog's hormone-free medium. Plantlets could be efficiently established in soil, thus confirming the possibility of biotechnological approaches with varieties of this crop species.Abbreviations E embryogenic - NE non embryogenic - MS Murashige and Skoog's (1962) medium - 2,4-D 2,4 dichlorophenoxyacetic acid - DAA days after anthesis - FWT fresh weight tissue     

Enhanced production and germination of somatic embryos by temporary starvation in tissue cultures of Daucus carota

Embryogenic callus was induced from cotyledonary explants of Daucus carota L. cultured on solidified MS medium supplemented with 1 mg l^-1 2,4-D. Following callus initiation somatic embryos were developed from the callus on MS medium without 2,4-dichlorophenoxyacetic acid. To stimulate the production and germination of somatic embryos we cultured the callus under physically and chemically modified conditions during subculture. When the embryogenic callus was cultured on half-strength MS medium or MS medium without sucrose or cultured under conditions of reduced humidity (69.3%), the production of embryos increased 3.4- to 4.5-fold compared to culture on MS medium containing 3% sucrose (control). Embryogenic callus cultured on MS medium after 5 days of starvation (by being placed in empty 12-well tissue culture plates) showed a 20-fold increase in somatic embryo production and enhanced maturation and germination of embryos. An important point is that the germination of somatic embryos with cup-shaped cotyledons, after a period in culture without medium, was remarkably improved (92%) compared to that of the controls (23%).Thus, we were able to show that stress by starvation without medium led to the enhanced production and increased germination of somatic embryos.     

Embryogenesis and plant regeneration from tissue culture of Canada wildrye,Elymus canadensis L.

Somatic embryogenesis and plant regeneration of Canada wildrye (Elymus canadensis L.) from tissue culture was investigated by culturing immature embryos and inflorescences on MS medium containing 2 mg/l 2,4-D. The optimum size of explants for maximum embryogenic callus formation was 1.0 to 1.5 mm for embryos and 4 to 6 cm for inflorescences. Plant regeneration from the subcultured embryogenic callus was attempted monthly using hormone-free MS medium or MS medium with 0.5 mg/1 2,4-D and 0.3 mg/l GA3. Three hundred and fifty seven plantlets were regenerated from the callus cultures of both explant sources during a six month period. Ten chlorophyll deficient plants accounting for 2.8% of the total regenerants were observed. One plant with white striped leaves survived and was found to be an octoploid.Abbreviations GA3 gibberellic acid - MS Murashige and Skoog (1962) - 2,4-D 2,4-dichlorophenoxyacetic acid     

Effects of auxin concentration on induction and growth of embryogenic callus from young inflorescence explants of Old World bluestem (Bothriochloa spp.) and bermuda (Cynodon spp.) grasses

Objectives of this research were to test the effects of plant genotypes and auxin 2,4-D (2,4-dichlorophenoxyacetic acid) medium concentrations on embryogenic (E) callus production of two grass species. Two Old World bluestem,Bothriochloa ischaemum, accessions (A-8793 and A-8911c) and three bermudagrass,Cynodon dactylon (L.) Pers., accessions (A-10978b, A12164, and Brazos) supplied the explant material. Immature inflorescences 9 mm in length were placed on modified Murashige-Skoog (MS) agar medium containing 0, 1, 3, or 5 mg L^-1 of 2,4-D. Explants of all genotypes produced callus by the end of a 4-week dark incubation period at 25°C. When subcultured onto fresh media and maintained at 25°C with a 16 hr photoperiod, calli became embryogenic within 8 weeks of inoculation. Three mg L^-1 of 2,4-D in the media maximized E callus production in both bluestem genotypes and in A-10978b and A-12164 bermudagrass genotypes. Maximum E callus production from Brazos bermudagrass resulted from the 1 mg L^-1 treatment. Somatic embryos developed after subculture under light. Embryos showed scutellum-like structures and coleoptile-coleorhiza bipolar organization. Plantlets were regenerated from all genotypes except Brazos, whose embryoids failed to germinate. All callus from Brazos eventually senesced. Light and scanning electron microscopy confirmed regeneration through somatic embryogenesis.     

Plant Regeneration Through Somatic Embryogenesis in Taxus wallichiana

We describe an efficient process for regeneration of Taxus wallichiana (Zucc) plants from callus cultures derived from zygotic embryos. Zygotic embryos cultured on half strength Lloyd and McCown’s basal medium supplemented with SH vitamin (1/2 WPMSH), 0.5 mg I^?1 6-benzyladenine (BA) and 1.0–2.0 mg I^?1 á-Napthaleneacetic acid (NAA) produced compact yellow (CY) callus after 4 weeks of culture. The 8-week-old CY call! (lines CY-A and CY-B) were initially slow growing but proliferated on transfer to WPM basal medium supplemented with 8.0 mg I^?1 2,4-D, 0.1–0.9 mg^?1 NAA and 0.3–1.0 mg^?1 BA after 4 weeks. Four morphologically distinct calli lines were obtained, of which only two call! lines, CY-B-FW and CY-B-FY were embryogenic. The 12-week-old callus line CY-B-FW developed globular somatic embryos on transfer to secondary medium after 8 weeks and matured in maturation medium after 4 weeks. Only 10% of the mature somatic embryos regenerated into complete plantlets after 4 weeks on conversion medium. Although the frequency of conversion was low, complete regenerated plantlets via somatic embryogenesis were obtained after 7–8 months of initiation of culture. Taxane analysis showed that the paclitaxel accumulation was higher in embryogenic callus than in non-embryogenic callus.     

Glycerol stimulation of chlorophyll synthesis,embryogenesis, and carboxylation and sucrose metabolism enzymes in nucellar callus of ‘Hamlin’ sweet orange

Anthers of niger (Guizotia abyssinica. Cass) were inoculated onto five different media differing mainly in their inorganic and organic constituents and plant growth regulators to study their influence on callus induction (embryogenic/non-embryogenic) and plant regeneration. LS medium supplemented with 2 mg 1^-1 2,4-d, and 0.3 mg 1^-1 KN favoured the production of EC, whereas 2 mg 1^-1 BAP and 0.5 mg 1^-1 KN promoted the NEC from anthers. Different types of embryos were initiated upon transfer of EC to Chaleff's R-2 medium containing 2 mg 1^-1 NAA and 0.3 mg 1^-1 KN and/or 5 mg 1^-1 ABA. NEC when transferred onto the medium supplemented with 1 mg 1^-1 BAP and 0.1 mg 1^-1 NAA produced on an average 8–12 shoots/callus mass. Embryoids developed from the EC and shoots differentiated from NEC when cultured onto the Chaleff's R-2 and MS media respectively lacking growth regulators, they transformed into whole plantlets. The plantlets thus obtained were successfully hardened and grown to maturity for analysis of various plant characters.Abbreviations EC embryogenic callus - NEC non-embryogenic callus - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - ABA abscisic acid - BAP 6-benzylaminopurine - KN kinetin - MS Murashige and Skoog's medium - LS Linsmaier and Skoog's medium     

High frequency somatic embryogenesis and synthetic seed production of the endangered species Swertia chirayita

An efficient protocol for plant regeneration through somatic embryogenesis was established from in vivo leaf explants of Swertia chirayita, a critically endangered medicinal herb. The highest frequency (76%) of embryogenic callus was induced on Murashige & Skoog (MS) medium supplemented with 0.5 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg/L kinetin (Kn) from in vivo leaf explants. Globular somatic embryos were induced and further matured from such embryogenic calli by subsequent culture on the same medium. The highest number of somatic embryos (48.83 ± 4.6) was recovered from embryogenic calli derived from leaf explants after 6 weeks of culture. Synthetic seeds were produced by encapsulating of torpedo stage embryos in sodium alginate (4% W/V) gel, dropped into 100 mM calcium chloride (CaCl₂ · 2H₂O) solution. The synthetic seeds were germinated on MS medium. The highest frequency of synthetic seed germination (84%) was observed on MS medium supplemented with 1.0 mg/L BA and 0.5 mg/L NAA. Regenerants were successfully acclimatized under ex vitro condition. This is the first report on synthetic seed production of S. chirayita. Application of these protocols would be helpful in reducing stress in natural habitat, and in long-term storage of elite genotypes through synthetic seed production.