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     

Plant regeneration through somatic embryogenesis from callus cultures of <Emphasis Type="Italic">Dioscorea zingiberensis</Emphasis>

A new method was established for somatic embryogenesis and plant regeneration from callus cultures of Dioscorea zingiberensis C.H. Wright. Primary callus was induced by culturing stems, leaves and petioles on Murashige and Skoog (MS) medium supplemented with 0.5–2.0 mg l^–1 N⁶-benzyladenine (BA) and 0–2.0 mg l^–1 -naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D) for 1 month. The highest frequency (87%) of callus formation was achieved from stem explants treated with 0.5 mg l^–1 BA and 2.0 mg l^–1 2,4-D. Somatic embryos were obtained by subculturing embryogenic calli derived from stem explants on MS medium supplemented with 2.0–4.0 mg l^–1 BA and 0–0.4 mg l^–1 NAA or 2,4-D for 3 weeks. The optimum combination of 4.0 mg l^–1 BA and 0.2 mg l^–1 NAA promoted embryo formation on one-third of the calli. After a further month of subculture on the same medium, mature embryos were transferred to MS medium supplemented with 0–4.0 mg l^–1 BA, NAA or indole-3-butyric acid (IBA) for further development of plantlets and tuber formation. Plant growth regulators had a negative effect on the development of mature embryos.     

Effects of ascorbic acid on axillary shoot induction in Tylophora indica (Burm. f.) Merrill.

A procedure for rapid in vitro multiplication of Tylophora indica (Burm. f.) Merrill., an important indigenous medicinal plant, has been developed. Addition of ascorbic acid was essential to induce sprouting of axillary buds. Optimum multiplication was observed on MS medium containing 6-benzylamino purine (5.0 mg l^–1), -naphathalene-acetic acid (0.5 mg l^–1) and ascorbic acid (100 mg l^–1). Rooting of in vitro produced shoots was readily achieved with indole-3-acetic acid alone (1.0 mg l^–1) in MS. The plantlets thus obtained were successfully transferred to pots in large numbers which grew normally.Abbreviations BAP 6-benzylamino purine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - 2ip 2-isopentenyladenine - Kn kinetin - MS Murashige & Skoog media - NAA -naphthalene acetic acid     

Effects of auxins and cytokinins on formation of Catharanthus roseus G. Don multiple shoots

The effects of different combinations of plant growth regulators and light intensity on the formation of multiple shoots of Catharanthus roseus (L.) were studied. By composing three dimension surfaces and their topo views from experimental data, it was clear that Murashige-Shoog (MS) medium supplemented with 7.0 mg l^-1 BA and 1.0 mg l^-1 NAA strongly stimulated the formation of shoots, whereas medium supplemented with 2,4-d suppressed the formation of shoots or caused shoot dedifferentiated. Light intensities of 550–700 Lux were found to be beneficial to the formation of shoots when MS medium was supplemented with 2 mg l^-1 6-BA and 0–1.0mg l^-1 NAA.Abbreviations BA-6 benzyladenine - NAA -naphthalenacetic acid - 2,4-d 2,4-dichlorophenoxyacetic acid     

Genetic transformation ofEustoma grandiflorum byAgrobacterium tumefaciens

Callus cultures were initiated from micropropagated Artemisia absinthium plantlets on MS basal medium supplemented with different concentrations of BA, Kn, NAA, IAA and 2,4-d in combination or singly. Supplementing the medium with low doses of both BA in combination with NAA, and Kn in combination with NAA enhanced the growth rate of callus cultures. However, cultures grew slowly following the second subculture and the majority turned brown and died within the next month. Initiation of root and shoot primordia occured directly from leaf explants cultured on 1.81 M 2,4-d, while adventitious shoot formation from callus was observed occasionally when BA was added to the medium in combination with IAA. Furthermore, medium containing 2.22 M BA and 2.69 M NAA stimulated both callus growth and organogenesis on some callus cultures derived from leaves and stems of young stock material. The best results were obtained with leaf explants. Cytological analysis of root meristems revealed that all regenerants were diploid (2n=18), as expected.Abbreviations MS Murashige & Skoog's salts and vitamins (1962) - BA 6-benzyladenine - NAA alphanaphthaleneacetic acid - Kn Kinetin (6-furfurylaminopurine) - 2,4-d 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid - FW fresh weight - Bi biomass increase     

Regeneration of niger (Guizotia abyssinica Cass.) CV Sahyadri from seedling explants

Root, hypocotyl and cotyledonary explants of niger (Guizotia abyssinica Cass) CV. Sahyadri were aseptically cultured on Murashige and Skoog's basal medium (MS) containing BAP and kinetin. Multiple shoot regeneration was induced from hypocotyl and cotyledonary explants while root explants produced only callus on MS medium supplemented with BAP. BAP (1 mg l^-1) was optimum for shoot regeneration. Regenerated shoots were transferred to MS medium without auxins, with auxins and with increasing concentrations of sucrose for rooting. Complete plantlets were obtained in all cases; however, 0.5 mg l^-1 NAA was the best for induction of roots. Ninety-seven per cent of the plantlets survived and completed their life cycle when transferred to natural conditions.Abbreviations BAP 6-benzylamino purine - NAA -naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid     

Rapid propagation of lemongrass (Cymbopogon flexuosus (Nees) Wats.) through somatic embryogenesis in vitro

Somatic embryos induced from callus cultures of lemongrass [Cymbopogon flexuosus (Nees) Wats.] on Murashige and Skoog medium supplemented with 5 mg/l of 2,4-D, 0.1 mg/l of NAA and 0.5 mg/l of Kn developed into plantlets when plated on a medium supplemented with 3 mg/l of BA, 1 mg/l of GA₃ and 0.1 mg/l of NAA. The regeneration potential of callus was retained for more than 2 years on the nutrient medium supplemented with comparatively lower levels of growth regulators (2,4-D at 2 mg/l, NAA at 0.1 mg/l and Kn at 0.25 mg/l). Approximately 30–35 plantlets were produced after two months of culture per 100 mg of callus inoculated. Regenerants were transplanted into soil and transferred to the field for assessment of various morphological and biochemical characteristics. The results of 1 year of field trials showed that plants derived from somatic embryoids were more uniform in all the characteristics examined when compared with the field performance of plants raised through slips by standard propagation procedures. Thus, a procedure has been developed for high frequency long term plant production of lemongrass through in vitro methods.Abbreviations 2,4-D 2,4 -dichlorophenoxyacetic acid - NAA -naphthalene acetic acid - Kn kinetin - BA benzyladenine - GA₃ gibberllic acid - MS Murashige and Skoog (1962) basal medium     

Plant regeneration from stem and petiole protoplasts of sweet potato (Ipomoea batatas) and its wild relative,I. lacunosa

A protoplast-to-plant regeneration system has been established for sweet potato (Ipomoea batatas (L.) Lam.) and its wild relative, I. lacunosa L. Viable protoplasts, isolated from preplasmolyzed stems and petioles of in vitro-grown plants, were cultured on liquid MS (Murashige & Skoog 1962) medium that supported cell division and colony formation. Embryogenic calli of sweet potato were induced on agar-solidified MS medium supplemented with 3% (w/v) sucrose, 50 mg l^-1 casamino acids, 0.2–0.5 mg l^-1 2,4-d, 1.0 mg l^-1 kinetin and 1.0 mg l^-1 ABA. On average, 3 plants were regenerated from a single sweet potato callus subcultured on semi-solid MS medium containing 3% (w/v) sucrose, 800 mg l^-1 glutamine, 2.0 mg l^-1 BA or 1.0 mg l^-1 kinetin and 1.0 mg l^-1 GA₃. Embryogenic calli of I. lacunosa L. were initiated on semi-solid MS medium containing 0.2–0.5 mg l^-1 IAA and 1.0–2.0 mg l^-1 BA. An average of 5 plants was regenerated from a single sweet potato callus subcultured on semi-solid MS medium containing 0.5 or 1.0 mg l^-1 GA₃.Abbreviations ABA abscisic acid - BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole acetic acid - MES 2-(N-morpholino)-ethane sulfonic acid - NAA -naphthaleneacetic acid     

Somatic embryogenesis and plant regeneration from callus cultures of Aconitum heterophyllum Wall

Plants were obtained via somatic embryogenesis in callus derived from in vitro raised leaf and petiole explants of Aconitum heterophyllum Wall. Callus was induced on a Murashige-Skoog medium supplemented with either 2,4-dichlorophenoxy acetic acid (2,4-d 1 mg l^-1) and kinetin (KN 0.5 mg l^-1) with coconut water (CW 10% v/v) or naphthalene acetic acid (NAA 5 mg l^-1) and benzylaminopurine (BAP 1 mg l^-1). Somatic embryos appeared after 2–3 months or 2 subculture passages when 2,4-d or NAA induced source of the callus was transferred to a MS medium containing BAP (1 mg l^-1) and NAA (0.1 mg l^-1). For successful plantlet formation, the somatic embryos were transferred to a medium containing 1/4 strength MS nutrient with indole-3-butyric acid (IBA 1 mg l^-1). Alternatively, the somatic embryos were dipped in a concentrated solution of IBA for 5 min and placed on a hormone free medium. Complete plantlets were formed after 4 weeks and were transferred successfully to soil.CIMAP Publication No. 1020.     

Plant regeneration from protoplasts isolated from cotyledons of Sesbania bispinosa

Protoplasts were isolated from cotyledons of Sesbania bispinosa (Jacq.) W.F. Wight. In a liquid-over-agar culture system with Murashige and Skoog (MS) medium supplemented with 1 mg l^-1 2,4-dichlorophenoxyacetic acid (2,4-d, 2 mg l^-1 benzyladenine (BA), 1 mg l^-1 glutamine and 0.5 and formed callus. The first division occurred after 3–4 days. Callus formed from the protoplasts differentiated shoots by organogenesis on MS medium with 1 mg l^-1 indolebutyric acid (IBA) and 1 mg l^-1 BA. These shoots developed into complete plantlets when excised and cultured on MS medium with 0.5 mg l^-1 IBA.     

The in vitro production of an anthocyanin from callus cultures of Oxalis linearis

Callus growth and the production of anthocyanins were sustained on the salts and vitamins of Murashige and Skoog. Callus growth was stimulated at a concentration of 8–32 M -naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-d). Benzyladenine (BA) and zeatin at 8 M inhibited callus growth whereas isopentenyladenine (iP) stimulated callus growth. NAA repressed anthocyanin production with an increase in NAA from 8–32 M. Anthocyanin synthesis was promoted by an increase in 2,4-d from 0.5 to 2 M and decreased thereafter up to a concentration 32 M 2,4-d. A concentration of 8 M BA, thidiazuron and zeatin, respectively stimulated pigment production. Sucrose stimulated callus growth at 60 mM and pigment production at 120–360 mM.Abbreviations BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - iP isopentenyladenine - TZ thidiazuron-N-phenyl-N-1,2,3-thiadiazol-5-yl-urea - Bu-HCl Butanol-2N HCl - BAW Butanol-acetic acid-water     

Taxus callus cultures: Initiation,growth optimization,characterization and taxol production

Callus was induced from Taxus baccata cv. Repandens Parsons ex Rehd., T. brevifolia Nutt., T. cuspidata Sieb. & Zucc., and T. x media cvs. Hicksii and Densiformis Rehd. using different concentrations of 2,4-d-(2,4-dichlorophenoxyacetic acid), IBA (indole-3-butyric acid), or NAA -naphthalene acetic acid in combination with kinetin. All cultures grew slowly following the first subculture, and a majority turned brown and ceased growth within the next six to twelve months. The callus cultures which lived, continued to grow very slowly for one to two years before the growth rate improved. Initiation of roots and shoot primordia-like structures occurred on some cultures maintained in the dark, and 16 h light/8 h dark, respectively. A fast-growing, habituated callus line (CR-1) derived from T. x media Rehd. cv. Hicksii was established from callus initiated in 1986. Supplementing the medium with casein hydrolysate and both fructose and glucose enhanced the growth rate. A great deal of heterogeneity was found among and within the callus, with respect to the amount of taxol produced. The callus exhibited levels of taxol ranging from 0.1 to 13.1 mg kg^-1 (0.0001 to 0.0131%) on a dry weight basis. Overall, the older brown-colored callus produced more taxol than the younger pale yellow-colored callus. The presence of taxol in callus samples was established by high performance liquid chromatography, its biological activity confirmed by a microtubule-stabilizing bioassay and its structure confirmed using one-and two-dimensional ¹H and ¹³C nuclear magnetic resonance spectroscopy.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - NAA -naphthaleneacetic acid - kinetin 6-furfurylaminopurine - 2iP 6-(,-dimethylallylamino)purine     

Somatic embryogenesis from leaf cultures of potato

An efficient procedure has been developed for inducing somatic embryogenesis from leaf cultures of potato cv. Jyothi. Leaf sections were initially cultured on 2,4-dichlorophenoxyacetic acid (2,4-D) + benzyladenine (BA) and -naphthaleneacetic acid (NAA) + BA supplemented Murashige and Skoog (MS) media. Nodular embryogenic callus developed from the cut ends of explants on media containing 2,4-D and BA, whereas compact callus developed on media containing NAA and BA. The explants with primary callus were subsequently moved onto MS media containing zeatin and/or gibberellic acid (GA₃) and BA. Treatment with zeatin (22.8 M) and BA (10.0 M) resulted in the induction of the highest number of somatic embryos directly from meristematic centres produced on the nodular tissue. Embryo induction and maturation took place on this medium. The cotyledonary stage embryos developed into complete plantlets on hormone-free MS medium. A distinct feature of this study is the induction of somatic embryogenesis in leaf cultures of potato which has not been reported previously.     

Embryogenesis and plant regeneration from anther culture of bamboo (Sinocalamus latiflora (Munro) McClure)

Embryogenic callus was initiated from bamboo (Sinocalumus satiflora (Munro) McClure) anthers cultured on N₆ medium supplemented with 1 mg/l 2,4-D, 1 mg/l BA, 2 g/l charcoal, 0.8% agar (Sigma) and 9% sucrose. Anthers with microspores at miduninucleate to early-binucleate stages showed better rate of response for callus induction. Prolonged culture of these embryogenic calli on the original medium or subculture to an auxin-free medium resulted in embryoid formation and their subsequent germination to form rooted plantlets. Chromosome counts from root-tip cells of anther-derived plant indicated that they were haploid (N=36).Abbreviations N₆ Chu et al. (1975) - MS Murashige and Skoog (1962) - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - BA 6-benzylaminopurine     

Rhizome induction and plantlet regeneration of Cymbidium goeringii from flower bud cultures in vitro

Apical flower buds of Cymbidium goeringli Reichenbach fil. (ca 2 mm long) exeised from infloreseences (ca 5 cm long) were explanted on modified Murashige & Skoog medium (=MS medium) supplemented with N⁶-benzyladenine (BA) and -naphthaleneacetic acid (NAA). Within 107 days of culture, swelling growth, chlorophyll synthesis, and subsequent rhizome differentiation were observed. MS medium containing 0.1 mg l^-1 BA and 10 mg l^-1 NAA was found to be optimal for initiating rhizome development and subsequent plantlet regeneration.Explants cultured on MS medium supplemented with 1 mg l^-1 NAA alone formed a mass of rhizome branches. Multiple shoots of rhizome branches were induced from apical segments when rhizomes were transferred to MS medium containing 0.1 mg l^-1 BA and 10 mg l^-1 NAA.Abbreviations NAA -naphthaleneacetic acid - BA N⁶-benzyladenine     

Regeneration of patchouli (Pogostemon cablin Benth.) plants from leaf and node callus, and evaluation after growth in the field

Pogostemon cablin (Benth.) is commercially important for its aromatic patchouli oil. Plants were regenerated through callus culture from leaf and nodal segments. Highest callusing was obtained from leaf explants in Murashige and Skoog (MS) medium supplemented with 3% sucrose, 0.8% bacto agar and 2 mg/L NAA + 0.5 mg/L BA. Shoot formation frequency was maximum (83%) with BA at 1.0 mg/L. Regenerated plantlets were rooted on MS medium with auxins. Maximum (90%) rooting was obtained using 0.5 mg/L NAA. Plantlets were grown for 4 weeks in this medium and then transferred to pots containing sterile sand in a moisture saturated glass chamber under laboratory conditions. The established plants were grown in pots filled with a mixture of sandsoilmanure (211) under natural daylight conditions in the field. The total leaf yield was increased in the tissue culture derived plants. These plants were dwarf and had higher specific leaf weight (leaf thickness) and leaf area compared to control plants.Abbreviations BA N⁶-Benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA Indole-3-butyric acid - IAA Indole 3-acetic acid - MS Murashige and Skoog (1962) medium - NAA 1-Naphthaleneacetic acid     

Rapid clonal propagation of Pinellia ternata by tissue culture

Adventitious buds or protocorm-like bodies were regenerated directly from excised explants without intervening callus. Differences in the ability of regeneration were observed among different plant organs with bulbils showing the highest regenerative ability followed by leaf blade and petiole. Ability of vegetative propagation of bulbil could be maintained by alternate solid-and liquid-medium culture. Theoretically, 1.7×10²⁷ plantlets could be produced from a single bulbil by this technique within one year based on the production and rapid growth of protocorm-like bodies and adventitious buds. Concentration of MS salts, NAA and sucrose influenced not only root formation from the differentiated adventitious buds, but also root number and length. For root formation, the best combination was one-half strength MS salts with 3–5% sucrose and 1 mg/l NAA. The high survival rate of 96% was recorded when plantlets were transplanted into a mixture of vermiculite:loam soil:peat moss (1:2:1). Plants from in vitro culture were morphological similar to field-grown plants. The acute toxicity of crude extracts from protocorm-like bodies was about one-fourth that of extracts from tubers of field-grown plants when tested with white mice. Tissue culture has potential for clonal propagation of Pinellia ternata plants for commercial use.Abbreviations MS Murashige and Skoog (1962) - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - BA 6-benzylaminopurine     

Tissue culture and micropropagation of Cuphea ericoides,a potential source of medium-chain fatty acids

Plant rgeneration occurred on leaf-and stem-derived callus of Cuphea ericoides Cham. & Schlechtd obtained in Murashige and Skoog medium supplemented with auxins [indole-3-acetic acid (IAA), -naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-d)] plus cytokinins [6-benzyladenine (BA) or kinetin]. These calluses were subcultured and showed vigorous growth. When subcultured on medium containing 2.22 or 4.44 M BA, the calluses showed profuse regeneration of shoots whereas those subcultured on medium supplemented with 2.69 M NAA or 0.226 M 2,4-d produced numerous roots. Isolated shoots rooted on Murashige and Skoog medium lacking growth regulators or containing 0.54 M NAA or 0.49 M indole-3-butyric acid (IBA). Plantlets were acclimatized to greenhouse conditions.Abbreviations BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige & Skoog medium - NAA 1--naphthaleneacetic acid     

Plant regeneration from callus and protoplasts in Medicago polymorpha

Seventeen ecotypes of the wild species Medicago polymorpha adapted to a Sardinian (Italy) environment have been evaluated for their response to tissue culture. The accession Samughero-Albi was the more respondent for callus induction and, together with Usassai, showed the highest regeneration capacity on media containing 1 mg l^-1 2iP and 0.1 mg l^-1 IAA. The morphogenetic response was also affected by the explant source. The hypocotyl-derived-calli were the best regenerating tissues. Regenerated plantlets were difficult to root and it was possible to obtain plants with a well developed root system only after 5–7 weeks of culture on media containing 2iP and IAA both at 0.2 mg l^-1. Mesophyll cells were the best protoplast yielding source but only those isolated from roots were able to divide and to regenerate plants. Results are discussed in relation to the genotype specificity for the morphogenetic response and the feasibility of using M. polymorpha in the somatic hybridization with M. sativa.Abbreviations NAA -naphthaleneacetic acid - 6-BAP 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - 2iP N⁶-²-isopentenyl-adenine - IAA indole-3-acetic acid - GA₃ gibberellic acid - GFMS growth regulator free MS medium - Prol proline - Malt maltose     

Rapid and high frequency shoot regeneration from hypocotyl protoplasts of Brassica nigra

Protoplasts, isolated from etiolated hypocotyls of seven day old seedlings of Brassica nigra, were cultured in Kao's liquid medium containing 7.2% glucose, 2,4-d (1 mg 1^-1), NAA (0.1 mg 1^-1) and zeatin riboside (0.5 mg 1^-1). After initial incubation for 3 days in dark at 25±1°C, cultures were transferred to a photoperiod cycle of 16/8 h and diluted on seventh and tenth day with MS medium containing 3.4% sucrose, 2,4-d (0.1 mg 1^-1) and BAP (1 mg 1^-1). About 62% of the cells divided at least once and 46% of them reached 8–16 cell stage in one week. The dividing cell clusters could be plated on agarose medium on the fifteenth day to obtain proliferating minicalli with a plating efficiency of 1.8%. 56.8% of minicalli, regenerated shoots on a regeneration medium containing 2 IP and IAA at 1 and 0.2 mg 1^-1 respectively. The in vitro produced shoots were rooted in MS medium containing 1 mg 1^-1 IBA and established in soil without difficulty. The time taken for protoplasts to develop into plants varied from 9 to 10 weeks.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - 2 IP 2-isopentenyladenine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - NAA -naphthaleneacetic acid - Kn kinetin