High frequency plant regeneration of Cymbopogon martinii (Roxb.) Wats by somatic embryogenesis and organogenesis

Embryogenic callus cultures were obtained upon repeated sub-culture of non-embryogenic callus from nodal segments of Cymbopogon martinii (Roxb.) Wats. Murashige and Skoog's medium supplemented with 1mg/l 2,4-dichlorophenoxyacetic acid and 0.5 mg/l kinetin and Linsmaier and Skoog's medium supplemented with 2mg/l 2,4-dichlorophenoxyacetic acid and 0.4 mg/l kinetin were used as maintenance media for non-embryogenic and embryogenic cultures, respectively. Plant regeneration occurred through organogenesis in MS basal media containing 2 mg/l kinetin, 1 mg/l 6-benzylaminopurine, 0.2 mg/l biotin, 0.2 mg/l Ca-pantothonate and 0.1 mg/l napthalene acetic acid. Embryogenesis was induced in LS medium supplemented with 1 mg/l kinetin, 0.5 mg/l 6-benzylaminopurine and 0.1 mg/l 3-indole acetic acid. Plant regeneration at high frequency was recorded both through organogenesis and embryogenesis in different passages of long term callus cultures.Abbreviation MS Murashige and Skoog medium - LS Linsmair and Skoog medium - BAP 6-benzylaminopurine - kin kinetin - 2,4-D 2,4-Dichlorophenoxyacetic acid - IAA Indole-3-acetic acid - CH Casein hydrolysate - CaP calcium pantothonate - NAA napthalene acetic acid     

Phenylacetic acid induced organogenesis in cultured leaf segments of Dianthus chinensis

Summary Shoot regeneration was achieved from leaf derived callus of Dianthus chinensis using Phenylacetic acid (PAA). Callus from basal leaf segments, raised on Murashige and Skoog's (MS) medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) or 1-Naphthaleneacetic acid (NAA) in combination with 6-benzylamino purine (BAP), was subcultured on medium supplemented with BAP in combination with 2,4-D, NAA or PAA. Shoots were induced only when leaf derived callus was subcultured on medium containing BAP (2.0, 5.0 mg/l) in combination with PAA (0.5, 1.0 mg/l). No shoot regeneration was observed when 2,4-D, NAA or BAP were used in the medium either singly or in different combinations. These results demonstrate that PAA in combination with BAP was essential to trigger shoot regeneration from cultured leaf callus of D. chinensis.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - DPX dibutylphthalate xylol - MS Murashige and Skoog (1962) basal medium - NAA 1-Naphthaleneacetic acid - PAA Phenylacetic acid     

Plant regeneration through somatic embryogenesis from mature seed and young inflorescence of wild rice (Oryza perennis Moench)

Somatic embryogenesis in the wild rice species (Oryza perennis) was induced from cultured mature seeds and young inflorescences. Murashige and Skoog's (MS) medium supplemented with 2 mg/l 2,4-D and 0.2 mg/l BAP was used for induction of a compact, white nodular callus and somatic embryos. Plant regeneration occurred with the tranfer of the nodular callus to MS basal medium containing 0.5 mg/l IAA, 0.5 mg/l NAA, 4 mg/l BAP and 500 mg/l casein hydrolysate. The embryogenic nature of the callus from both explants was maintained over 10 subcultures for about 12 months. Plant regeneration with respect to the number of calli plated from the 6th to 10th passage varied from 80% to 60% for young inflorescence derived callus and from 75% to 69.8% for seed-derived callus.Abbreviations MS Murashige and Skoog medium - BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA naphthalene acetic acid - CH casein hydrolysate     

Regeneration of plantlets from the callus of stem segments of adult plants of Ficus religiosa L.

Stem segments of adult plants of Ficus religiosa L. cultured on MS medium containing 1.0 mg/l 2,4-D produced callus. Shoots were regenerated when the induced calli were transferred to medium supplemented with 0.05 to 2.0 mg/l BAP. Callus derived shoots produced roots and developed into plantlets when transferred to medium supplemented with 1.0 mg/l NAA.Abbreviations MS Murashige and Skoog (1962) - BAP 6-benzylaminopurine - NAA naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Somatic embryogenesis and plant regeneration from cell suspension and tissue cultures of mature himalayan poplar (Populus ciliata)

Somatic embryogenesis and plantlet formation were obtained from callus and cell suspension cultures of 40-year- old Himalayan Poplar (Populus ciliata Wall ex Royle). Callus and cell suspensions were obtained by transfer of inoculum of semiorganized leaf cultures, which were maintained on Murashige and Skoog (MS) medium supplemented with benzylaminopurine (BAP), to MS with 2,4-dichlorophenoxyacetic acid (2,4-D). Reduction of 2,4-D concentration during subsequent subculture of cell suspensions resulted in the formation of embryoids. These embryoids developed further only after being transferred to agar-based MS medium supplemented with BAP and naphthalene acetic acid. Loss of embryogenic potential was observed in cell suspensions after 6 subcultures. However, callus cultures retained the embryogenic potential even after repeated subcultures for more than a year. Plantlets could be successfully hardened and grown in natural outdoor conditions.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthalene acetic acid - MS Murashige and Skoog (1962) medium     

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     

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 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     

Somatic embryogenesis inGnetum ula Brongn. (Gnetum edule) (Willd) Blume

Summary Somatic embryos ofGnetum ula (Gnetum edule) an endangered gymnosperm closely related to the angiosperms have been induced in vitro. Megagametophyte tissue with immature embryos was cultured on Murashige and Skoog medium. A mucilaginous, translucent embryogenic callus was obtained with 5 mg/l BA. Callus induced with 2,4-D was non-embryogenic. The embryogenic callus in liquid half strength Murashige and Skoog medium without inorganic nitrates supplemented with 2.5 g/l casein hydrolysate and 0.5 g/l L-glutamine gave rise to immature embryos. The embryos matured when treated with 60 g/l sucrose and 10 mg/l abscisic acid.Abbreviations MS Murashige and Skoog - BA 6-benzylaminopurine - 2,4-D 2,4 - dichlorophenoxyacetic acid - ABA abscisic acid     

High-frequency plant regeneration from coleoptile tissue of indica rice (Oryza sativa L.)

Summary An efficient method was established for high-frequency embryogenic callus induction and plant regeneration from 3-,4-, 5- and 7-d-old coleoptile segments of Indica rice (Oryza sativa L. cv. Kasturi), Compact and friable callus developed from the cut ends and also on the entire length of the coleoptile segments cultured on Murashige and Skoog (MS) basal medium (1962) supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D, 4.50–18.0 μM), kinetin (2.32 μM) and sucrose (3%, w/v). High frequency embryogenic callus induction and somatic embryo development was achieved when embryogenic calluses were transferred to MS medium supplemented with 2.25 μM 2,4-D, 2.32 μM kinetin, 490 μM L-tryptophan and 3% (w/v) sucrose. Plant regeneration was achieved by transferring clumps of embryogenic callus onto MS medium containing 2.85 μM indole-3-acetic acid (IAA), 17.77 μM 6-benzylaminopurine (BA) and 3% (w/v) sucrose. Histological observations of embryogenic calluses revealed the presence of somatic embryos and also plant regeneration via multiple shoot bud formation. Three, 4- and 5-d-old coleoptile segments showed a significantly (P<0.05) higher frequency of plant regeneration and mean number of plantlets per explant in comparison to 7-d-old coleoptile segments. The highest frequency (73.5%) of plant regeneration and mean number of plantlets (11.9±1.0) was obtained from 4-d-old coleoptile segments. Regenerated shoots were rooted on MS basal medium containing 4.92 μM indole-3-butyric acid (IBA) and plants were successfully transferred to soil and grown to maturity.     

Plant regeneration through somatic embryogenesis from suspension cultures of a minor millet,Paspalum scrobiculatum

Compact, friable and embryogenic calli were initiated from immature inflorescences and young leaf bases of one week old seedlings of Paspalum scrobiculatum cultured on MS medium supplemented with 2,4-D. A stable, embryogenic suspension culture was initiated from these calli and maintained in a liquid version of the same MS medium. Embryogenic calli and somatic embryos were obtained by plating suspension culture cells onto semi-solid medium containing 2,4-D. Complete, normal plantlets developed on 2,4-D free medium at a high frequency from somatic embryos. NAA and BAP in the medium promoted plant development.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - BAP 6-benzylaminopurine - ABA Abscisic acid - MS Murashige and Skoog (1962) - CM Coconut milk     

An efficient and rapid regeneration via multiple shoot induction from mature seed derived embryogenic and organogenic callus of Indian maize (Zea mays L.)

An efficient method for in vitro micro propagation and genetic transformation of plants are crucial for both basic and applied research. Maize is one of the most important cereal crops around the world. Regeneration from immature embryo is hampered due to its unavailability round the year. On the contrary mature embryo especially tropical maize is recalcitrant toward tissue culture. Here we report a highly efficient regeneration (90%) system for maize by using 2 different approaches i.e., embryogenic and organogenic callus cultures. Seeds were germinated on MS medium supplemented with 5 mg/l 2,4-D and 3 mg/l BAP. Nodal regions of 2 wks old seedlings were longitudinally split upon isolation and subsequently placed on callus initiation medium. The maximum frequency of embryogenic callus formation (90%) was obtained on MS medium supplemented with 2 mg/l 2,4-D and 1 mg/l BAP in the dark conditions. The compact granular organogenic callus formation (85% frequency) was obtained on MS medium supplemented with 2.5 mg/l 2,4-D and 1.5 mg/l BAP at light conditions. MS medium supplemented with 2 mg/l BAP, 1 mg/l Kinetin and 0.5 mg/l NAA promoted the highest frequency of shoot induction. The highest frequency of root formation was observed when shoots were grown on MS medium. The regenerated plants were successfully hardened in earthen pots after adequate acclimatization. The important advantage of this improved method is shortening of regeneration time by providing an efficient and rapid regeneration tool for obtaining more stable transformants from mature seeds of Indian tropical maize cultivar (HQPM-1).     

Plantlets from somatic callus tissue of the woody legume Sesbania bispinosa (Jacq.) W. F. Wight

In vitro regeneration of plantlets and multiplication of Sesbania bispinosa (Jacq.) W.F. Wight plants from cultured callus tissue were demonstrated. Callus was established from both cotyledons and mature leaflets on Murashige and Skoog (MS) basal medium supplemented with BAP (0.5 mg/l) and 2,4-D (2 mg/l). Callus mediated shoot bud differentiation was studied under defined nutritional, hormonal and cultural conditions. Various concentrations of BAP or kinetin (Kn) with coconut milk (CM) in MS media induced different levels of shoot bud differentiation as well as multiplication. Multiple shoot bud differentiation occurred in most of the primary calli. The best medium for shoot bud differentiation from cotyledon derived callus, contained BAP (2 mg/l) and 15% CM (V/V). More efficient shoot bud organogenesis was recorded with BAP than Kn. Supplementation with CM in MS media accelerated shoot bud organogenesis in differentiating callus tissue. Rooting of differentiated shoots was achieved by a three step culture procedure involving (a) MS solid medium containing IBA (2 mg/l), (b) growth regulator free half strength MS medium with 1% charcoal, and (c) half strength MS liquid medium free of vitamins, growth regulators and charcoal.Abbreviations IAA indoleacetic acid - IBA indole-3-butyric acid - NAA naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - Kn kinetin - CM coconut milk - MS Murashige and Skoog's medium - SBI shoot bud inducing medium     

Effect of abscisic acid and callus size on regeneration of american and international rice varieties

Embryogenesis and plant regeneration of Texas and international rice, Oryza saliva (L.), varieties (both indica and japonica types) were induced in culture on a regime consisting of the use of ABA or BAP in the subculture medium and small (10 mg) callus pieces on the regeneration medium. Ten 10 mg callus pieces on regeneration medium resulted in a 2- to 10-fold increase in plant regeneration over single 100 mg pieces. Plant regeneration of Texas rice cultivars (Lemont, Rico I, Rexmont and Skybonnet) and Taipei 309 was enhanced by the use of ABA in the subculture medium with a 2-fold and a 3- to 10-fold increase in plant regeneration with 2.6 mgL^–1 and 26 mgL^–1 ABA in the subculture media, respectively. Regeneration of plants from callus of IR36 and IR64 was not enhanced by ABA but by the use of BAP and Trp in the subculture medium or by 2,4-D alone. The subculture medium containing BAP and Trp produced a 5-fold increase in plant regeneration rate from IR64 callus and was equal to subculture medium containing only 2,4-D for IR36 callus. Both Lemont and IR36 were previously reported to be difficult to regenerate or non-regenerative, however, the use of ABA or BAP in the subculture medium, small callus pieces and visual selection of embryogenic callus allowed the regeneration of up to 20 and 22 plants from 100 mg of Lemont and IR36 callus, respectively.Abbreviations ABA abscisic acid - BAP benzylaminopurine - MS Murashige and Skoog - NAA napthaleneacetic acid - Trp tryptophan - 2,4-D 2,4-dichlorophenoxyacetic acid     

Plant regeneration through somatic embryogenesis from callus induced on immature embryos of Alstroemeria spp. L.

Summary The plant regeneration ability of callus obtained from zygotic embryos of the monocot Alstroemeria spp. was studied. The best explants for somatic embryogenesis were immature zygotic embryos in half-ovules when the endosperm was still soft and white. For 2 genotypes embryogenic callus was induced on callus induction medium with a success rate of 54%. The best callus induction period was 10 weeks. The morphology of embryogenic callus was nodular. Somatic embryos were formed after transfer of the callus to regeneration medium. These somatic embryos revealed later on the typical features of zygotic Alstroemeria embryos. The total duration of the plant regeneration protocol, from inoculation till rooted plantlets ready for transfer to the greenhouse, was 28 weeks.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) - NAA -naphthaleneacetic acid     

Rapid mass propagation of Chrysanthemum morifolium by callus derived from stem and leaf explants

A procedure for rapid multiplication of Chrysanthemum morifolium RAMAT cv. Birbal Sahni using leaf callus and stem (nodal/internodal) callus as well as node and apical shoots has been developed. Murashige and Skoog's medium (1962) supplemented with 2mg/1 2,4-D yielded good green calli from both leaf and stem segments within 2 weeks. About 1 cm × 1 cm callus regenerated 2–3 shoots after 3 weeks on MS solid medium supplemented with 0.1 mg/l IAA and 0.2 mg/l BAP. Each of the regenerated shoots when transferred to the same shooting medium without agar yielded about 150 new shoots, which in turn regenerated roots after another week in MS half strength or modified White's media (Rangaswamy, 1961). It has been estimated that about 10¹⁴ plantlets could be produced in a year from one expiant following the proposed protocol.Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog's (1962) medium     

Somatic embryogenesis and plant regeneration in cultured immature inflorescences of Setaria italica

Young inflorescence explants of Setaria italica in culture showed high capacity for regenerating plantlets through somatic embryogenesis. Embryogenic callus formation was initiated from the explants cultured on Murashige and Skoog's medium with 2 mg/l 2,4-D and 0.2–0.5 mg/l KT or BAP, but it was better for the maintenance of embryogenic growth to subculture the calli on the medium with 2,4-D and KT/BAP and on the medium with 2 mg/l 2iPA and 0.2 mg/l NAA alternately. A number of plantlets were regenerated when embryogenic calli were transferred onto the same basic medium but with 2 mg/l BAP and 0.5 mg/l NAA. Plant regeneration capacity has been maintained in some embryogenic calli during fourteen months of subculture.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - IAA 3-indoleacetic acid - 2iPA N⁶-(²-isopentenyl) adenosine - BAP 6-benzylaminopurine - KT kinetin - CH casein hydrolysate     

Somatic embryogenesis and plant regeneration of squash Cucurbita pepo L cv. YC 60

Plant regeneration from tissue cultures of summer squash, Cucurbita pepo L., cv. YC60, has been observed. Somatic embryos organized from shoot apex derived callus cultured on Murashige and Skoog (MS) medium supplemented with 1.2 mg/l 2,4,5-trichlorophenoxyacetic acid, 0.8 mg/l benzylaminopurine, and 0.1 mg/l kinetin. Embryos developed into plantlets by transfer of immature somatic embryos to MS medium with 0.05 mg/l NAA and 0.05 mg/l kinetin. Regenerated plants appeared morphologically normal and set fruits with seeds which could germinate normally.Abbreviation BAP 6-benzylaminopurine - 2,4-D 2, 4dichlorophenoxyacetic acid - IAA indole-3-acetic acid - KN kinetin - NAA -naphthyleneacetic acid - MS Murashige and Skoog - 2,4,5-T 2, 4,5-trichlorophenoxyacetic acid     

Plant regeneration from callus cultures of Valeriana wallichii DC.

Petiole expiants of Valeriana wallichii. DC., a threatened medicinal plant, were used for inducing callus. Optimum callus formation was observed on Murashige and Skoogs' (1962) medium supplemented with 3.0 mg/l NAA and 0.25 mg/l Kn. Shoot regeneration was achieved upon transferring the callus to medium containing 1.0 mg/l Kn and 0.25 mg/l NAA. Complete plantlets were obtained on the same medium or upon transfer of the regenerated shoot buds to medium containing 5.0 mg/l Kn and 1.0 mg/l IAA. Nearly a thousand callus regenerated plants were successfully transferred to the field following previously standardized hardening procedures.Abbreviations BAP 6-Benzylaminopurine - 2,4-D 2,4-dichloro phenoxyaceticacid - 2iP 2-isopentenyladenine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - Kn Kinetin - MS Murashige and Skoog's medium (1962) - NAA -napthalene aceticacid - Z Zeatin     

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