Regeneration in cultures of Papaver bracteatum as influenced by growth hormones and temperature

Callus was induced on Papaver bracteatum Lindl. seedlings inoculated on Murashige & Skoog (1962) medium supplemented with -naphthaleneacetic acid (NAA) (1.0 mg 1^-1) and benzylamine purine (BA) (0.5 mg 1^-1). Subculture resulted in excellent callus proliferation but no organogenesis. Shoots were regenerated in cultures grown on MS medium containing NAA (1.0 mg 1^–1), BA (0.5 mg 1^–1) and casein hyrdrolyzate (2.0 mg 1^-1). The shoots developed into plantlets after 8 weeks of culture, and were induced to root on 1/2 MS without the addition of growth regulators. The rooted plantlets were transferred to soil after hardening.MS at full strength was found inhibitory for callus induction and proliferation, but 1/2 MS was suitable. Similarly callus growth was very slow at 25°C but increased when the temperature was lowered to 15°C as did bud initiation.Abbreviations BA benzylamine purine - IAA indoleacetic acid - K kinetin (6-furfurylamino purine) - NAA -naphthalene acetic acid - 2,4-d 2,4-dichlorophenoxyacetic acid     

One step shoot tip multiplication and rooting of Digitalis thapsi L.

Shoot tips from seedlings of Digitalis thapsi L. were cultured on Murashige and Skoog's medium and the effect of various auxins (2,4-D, NAA and IAA) were analyzed alone or in combination with cytokinis (BA and kinetin). Shoot multiplication and direct rooting of the new shoots were obtained after four weeks of culture in MS medium without hormones, but callus formation and the appearance of abnormal phenotypes were frequent. The addition of auxins to the cultures prevented the formation of callus but not the appearance of variant phenotypes. Both drawbacks could be avoided by combination of NAA or IAA with BA or kinetin. The best results for shoot multiplication and direct rooting were obtained with 0.5 mg l^-1 NAA and 0.1 or 0.5 mg l^-1 kinetin.Abbreviations BA 6-benciladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - Kin kinetin - NAA naphtalene acetic acid - MS Murashige and Skoog     

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     

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     

Plant regeneration in Kentucky bluegrass (Poa pratensis L.) via coleoptile tissue cultures

Summary Plant regeneration in Kentucky bluegrass (Poa pratensis L. cv. Touchdown) via culture of seedling tissues was investigated. When coleoptile, leaf, and stem sections of dark-germinated seedlings were cultured on Murashige and Skoog (MS) medium, different types of callus were produced, depending on the expiant source and growth regulator combinations. Only compact-friable callus (type 3) and moderately compact, friable callus (type 2) produced shoots upon subculture. The nonstructured watery callus (type 4) produced roots without shoots. Shoot differentiation from callus tissues was highest when the culture medium contained 0.2 mgL^–1 picloram + 0.01 mgL^–1 -naphthaleneacetic acid (NAA). Calli grown from coleoptiles had higher shoot regeneration frequency (32%) than that obtained from either stem sections (12%) or young leaf tissues (2%) of the same seedlings. Some organogenic callus lines produced exclusively green plants, while others produced albino shoots or a mixture of green and albino shoots. The green plants were multiplied in a medium containing 0.1 mgL^–1 BAP plus either 0.2 mgL^–1 picloram or 0.1 mgL^–1 indole-3-acetic acid (IAA). Over 90% of the cultures in the shoot proliferation medium produced roots in 4 weeks. The rooted plants were successfully established in soil medium and grown in the greenhouse.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - MS Murashige and Skoog (1962) medium - NAA -naphthaleneacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid - TDZ thidiazuron     

Organogenesis, shoot regeneration, and flowering response of Vernonia cinerea to different auxin/cytokinin combinations

Summary A viable protocol has been developed for direct and indirect shoot regeneration of Vernonia cinerea. To establish a stable and high-frequency plant regeneration system, leaf and stem explants were tested with different combinations of α-naphthalene acetic acid (NAA), indole-3-acetic acid (IAA), and benzylaminopurine (BA). Lateral buds on nodal explants grew into shoots within 2 wk of culture in Murashige and Skoog (MS) basal medium supplemented with 20.9 μM BA. Excision and culture of nodal segments from in vitro-raised shoots on fresh medium with the same concentration of BA facilitated development of more than 15 shoots per node. Similarly leaf, nodal, and internodal explants were cultured on MS basal medium supplemented with different concentrations of BA, NAA, and IAA either alone or in combinations for callus induction and organogenesis. Shoot buds and/or roots were regenerated on callus. Shoot buds formed multiple shoots within 4 wk after incubation in induction medium. Adventitious buds and shoots proliferated when callus was cut into pieces and subcultured on MS basal medium containing 20.9 μM BA and 5.3 μM NAA. This combination proved to be the best medium for enhanced adventitious shoot bud multiplication, generating a maximum of 50 shoots in 4 wk. This medium was also used successfully for shoot proliferation in liquid medium. Root formation was observed from callus induced in medium containing 8.05–13.4 μM NAA. Regenerated shoots exhibited flowering and root formation in MS basal medium without any growth regulators. Plantlets established in the field showed 85% survival and exhibited identical morphological characteristics as the donor plant.     

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     

Effect of genotype,explant type and growth regulators on organogenesis in Morus alba

Plantlets of the mulberry (Morus alba L. vars. Chinese White, and Kokuso-27) were produced from callus cultures. For callus induction, leaf, internodal segments, and petiole explants of Chinese White, Kokuso-27 and Ichinose varieties were grown on MS basal medium fortified with 2,4-D and 6-benzylaminopurine (BA). Callogenesis was dependent on the nature of explant used, the genotype and growth regulators supplemented in the medium. Leaves were the best explant type used for callus induction. Best callogenesis was obtained on MS medium containing a combination of 1 mg l⁻¹ 2,4-D and 0.5 mg l⁻¹ BA (95-100%). Calluses formed shoots on MS medium supplemented with 1 mg l⁻¹ BA. Supplementation with 0.1 mg l⁻¹ 2,3,5-triiodobenzoic acid (TIBA) in this medium enhanced shooting response. Presence of TIBA in the medium also improved the long-term organogenic potential of the callus. Regenerated shoots produced roots on Murashige & Skoog (MS) medium containing either 0.5 mg l⁻¹ indole-3-butyric acid (IBA) or α-naphthaleneacetic acid (NAA). Seventy percent of the rooted plants were established in the field where they are performing well. This revised version was published online in June 2006 with corrections to the Cover Date.     

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     

Regeneration of fertile plants from protoplasts of sunflower (Helianthus annuus L.)

Summary Sunflower hypocotyl protoplasts (Helianthus annuus L.) from 5 PIONEER genotypes (PT024, SMF3, EMIL, HA300*PT024, VK5F) and 1 public line (RHa 274) formed colonies at frequencies of up to 60% when plated in 0.25ml agarose beads in a modified L4 medium (Lenée and Chupeau 1986) containing 3mg/l NAA, 1mg/l BA and 0.1mg/l 2,4-D, and 1000mg/l casamino acids. Protoplast-derived colonies grew slowly into calli. Organogenesis was obtained from callus of PT024 on a MS medium containing NAA and BA at 1mg/l and GA at 0.1mg/l. Freshly excised shoots were induced to root by an IAA treatment. Regenerated plants were transferred to the greenhouse and seed was harvested within 7 months of the initial protoplast isolation.Abbreviations BA 6-benzylaminopurine - NAA -naphtaleneacetic acid - GA gibberellic acid - IAA indole-3-acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog mineral elements - B5 Gamborg mineral elements     

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     

In vitro propagation of prairie gentian

Explants of shoot tips, internodal stem sections, and leaf segments of Lisianthus, Eustoma grandiflorum (Griseb.) Schinners, Dwarf Purple were cultured in vitro on modified Murashige and Skoog (MS) media. Explants of shoot tips and internodal stem sections developed into multiple shoots, whereas, leaf segments turned chlorotic on a medium supplemented with 3 mgl^-1 benzyladenine (BA) and 0.2 mgl^-1 naphthalene acetic acid (NAA). Shoot proliferation was obtained on shoot tips and leaf segments with 3 mgl^-1 BA, but internodal stem sections became necrotic and died on this medium. Rooting was induced in cultures with multiple shoots by subculturing explants on a half-strength MS medium supplemented with 2 mgl^-1 indole-3-acetic acid (IAA). Rooted plantlets were successfully transferred to soil.     

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     

Factors effecting adventitious shoot regeneration from leaf explants of quince (Cydonia oblonga)

A procedure for adventitious shoot regeneration from leaf explants of quince (Cydonia oblonga Mill.) using thidiazuron (TDZ) was developed. Excised leaves of cultures grown on Murashige and Skoog (MS) medium containing 5 M benzyladenine (BA) and 0.9% Gibco Phytagar were used. Several experiments were conducted to determine optimum concentrations of thidiazuron, -naphthaleneacetic acid (NAA) and sucrose. When the medium contained 1.5 M TDZ and 2.5 M NAA, 85% of the discs regenerated shoots with an average of eight shoots per leaf disc. An incubation period of three weeks in the dark was necessary for optimum shoot regeneration. Leaves excised from four to six-week-old cultures gave a higher percent shoot regeneration than leaves from cultures older than six weeks. Regeneration percentages were significantly reduced when sucrose concentration in the medium was less than 3%. A significantly higher percentage of shoots regenerated when leaf discs were placed on the regeneration medium abaxial side down as compared to the adaxial side.Regenerated shoots were cultured on MS medium containing 5 M BA and rooted on half-strength MS medium containing 10 M NAA. Rooted plantlets were acclimatized to greenhouse conditions for evaluation of any somaclonal variation. The importance of these findings are discussed in relation to in vitro improvement of plants.Abbreviations BA benzyladenine - MS Murashige & Skoog (1962) salt mixture - NAA -naphthaleneacetic acid - TDZ thidiazuron (N-phenyl-N'-1,2,3,-thiadiazol-5-ylurea) Approved for publication by the Director, West Virginia Agric. and For. Expt. Sta. as Scientific Article No.2346     

Rapid in vitro multiplication of jujube through mature stem explants

Stem explants obtained from a mature tree of Ziziphus mauritiana Lamk were grown on modified Murashige and Skoog medium containing 3800 mg l^-1 potassium nitrate, 2475 mg l^-1 ammonium nitrate, 11 M benzyladenine and 0.5 M indole-3-acetic acid. During successive subcultures 15–20 shoots per inoculum were produced. Rooting was induced by pretreatment with 50 M indolebutyric acid or 1-naphthaleneacetic acid for 24 h followed by transfer to auxin-free White's medium. Plantlets grew well in a soil and vermiculite mixture.Abbreviations IAA Indole-3-acetic acid - NAA 1-naphthaleneacetic acid - BA benzyladenine - MS Murashige and Skoog     

Direct shoot regeneration from leaf explants of <Emphasis Type="Italic">Spilanthes acmella</Emphasis>

Multiple shoots of Spilanthes acmella Murr. were induced from nodal buds of in vivo and in vitro seedlings on Murashige and Skoog (MS) medium containing 1.0 mg dm⁻³ 6-benzyladenine (BA) and 0.1 mg dm⁻³ α-naphthalene-acetic acid (NAA). Adventitious shoots were successfully regenerated from the leaf explants derived from the above mentioned multiple shoots. The efficiency of shoot regeneration was tested in the MS medium containing BA, kinetin, or 2-isopentenyl adenine in combination with NAA, indole-3-acetic acid (IAA), or indole-3-butyric acid (IBA) and gibberellic acid. Maximum number of shoots per explant (20 ± 0.47) was recorded with 3.0 mg dm⁻³ BA and 1.0 mg dm⁻³ IAA. An anatomical study confirmed shoot regeneration via direct organogenesis. About 95 % of the in vitro shoots developed roots after transfer to half strength MS medium containing 1.0 mg dm⁻³ IBA. 95 % of the plantlets were successfully acclimatized and established in soil. The transplanted plantlets showed normal flowering without any morphological variation.     

A simple and efficient plant regeneration system for leaf protoplasts ofNierembergia repens by inducing single shoots on the microcolonies

A simple and efficient protocol for plant regeneration from mesophyll protoplasts ofNierembergia repens is described. The protoplasts divided in modified half-strength MS (/12 MS) medium containing benzylaminopurine (BA) and a-naphthaleneacetic acid (NAA) and formed visible colonies after 2 weeks which produced single adventitious shoots 4 weeks later. Plating efficiency (11.2%), percent colony formation (0.84%), and the number of shoot-forming colonies (368/dish) were highest in /12 MS containing 0.1 mg/l BA and 0.05 mg/l NAA. However, the percentage of colonies with shoot formation was highest (31.8%) in /12 containing 0.05 mg/l BA and 0.01 mg/l NAA. Almost all of the remaining colonies (97.5%) also regenerated shoots upon transfer onto MS medium containing 0.05 mg/l BA. The shoots with 2–3 leaves readily rooted 3–5 days after insertion in /12MS lacking plant growth regulators. Regenerated plantlets were easily established in soil 50 days after protoplast isolation. All the regenerants were normal and possessed diploid chromosome numbers.Abbreviations BA Benzylaminopurine - FDA fluorescein diacetate - MES 2-(Morpholino)ethanesulfonic acid - MS Murashige and Skoog (1962) medium - /12MS half strength MS - NAA -naphthaleneacetic acid - PGR plant growth regulator     

In vitro adventitious shoot formation from leaf cultures of Clerodendrum inerme (L) Gaertn.

In vitro adventitious shoots (about 28) of Clerodendrum inerme were regenerated from leaf segments on MS medium containing BA (4 mg/L). These shoots developed directly from the leaf explants without callusing after 5 weeks. Leaf explant when cultured in MS medium containing BA (2 mg/L) and NAA (0.5 mg/L) developed compact callus that became nodular and regenerated shoots (about 50) after 5 weeks. The in vitro developed shoots were rooted in MS medium supplemented with IAA (2 mg/L). The hardened plantlets were successfully established in the field with 90% survival.     

Acquisition of competence for shoot regeneration in leaf discs ofSaintpaufla ionantha xconfusa hybrids (African violet) cultured in vitro

Leaf discs fromSaintpaulia ionantha xconfusa hybrids (African violet) were transferred between basal medium (BM) containing no hormones and shoot-inducing medium (SIM) containing 2.0 mg 1^–1 indole acetic acid and 0.08 mg l^–1 6-benzylaminopurine to determine whether there is a window of competence for shoot regeneration. Leaf discs precultured on BM prior to transfer to SIM formed buds 3 days earlier than the controls (leaf discs not precultured) regardless of whether the discs were placed upside down or right side up on the medium. This suggests that cultured leaf cells were not competent for shoot induction during the first 3 days of culture. Leaf discs cultured right side up (abaxial surface to the medium) did not form buds on BM alone, unlike discs cultured upside down. Leaf disc survival was affected by a delay in hormonal exposure, but surviving leaf discs produced as many shoots as control leaf discs. This suggests that in the absence of exogenous plant hormones, cellular competence to regenerate shoots is not lost in excised leaf discs of African violet.Abbreviations BM Basal medium - SIM_Sho Shoot-inducing medium     

Clonal propagation byin vitro culture of Corchorus (jute)

Callus was produced on cotyledon, shoot tip, hypocotyl and root explants of twoCorchorus species on several media. Cytokinin was necessary for callus production on cotyledon explants. BothC.olitorius genotypes produced most callus on media with zeatin and either NAA or IAA, and theC.capsularis genotype produced most callus on media with IAA and either zeatin or BA. High frequencies of regenerated shoots were obtained from shoot tip explants of both species, from the apical meristem and from callus. Media with 2.0 mg 1⁻¹ BA were superior for both species, and media with zeatin were equally good forC.capsularis only. More regeneration was obtained for all genotypes after subculture of callus on media with 2.0 mg 1⁻¹ zeatin. Cotyledon callus produced less regeneration, also with differences between genotypes; explants of both genotypes ofC.olitorius produced regeneration on a medium with NAA and zeatin, and theC.capsularis genotype produced regeneration on a medium with IAA and BA. Limited regeneration from root explant callus was obtained forC.capsularis only on medium with BA and IAA. Regeneration was not obtained from hypocotyl callus. Further regeneration of shoots of both species was obtained from secondary callus after subculture, and from nodal segments of regenerated shoots and of seedling shoots cultured on basic MS medium without growth hormones. Roots were produced on about 80% of all shoots after transference to medium with 0.2 mg 1⁻¹ IBA, and rooted plantlets survived and flowered normally after transference to compost.