Plant regeneration through direct shoot bud formation from leaf cultures of Paphiopedilum orchids

Leaf explants of Paphiopedilum phiIippinense hybrids (hybrid PH59 and PH60) directly formed adventitious shoots from wound regions within 1 month, when cultured on modified Murashige and Skoog medium (1/2-strength macro- and full-strength micro-elements) free of plant growth regulator in darkness. The combinations of 2,4-dichlorophenoxyacetic acid ((2,4-D) acid (0, 4.52 and 45.25 M) and 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea (TDZ) (0, 0.45, 4.54 and 22.71 M) were used to test their effects on direct shoot bud formation from two types of explants (1.5-cm long intact leaf explants and 0.5-cm long leaf segment explants). In hybrid PH59, 4.54 M TDZ increased mean numbers of shoots per explant with leaf segment explants. In hybrid PH60, 4.52 M 2,4-D plus 0.45 M TDZ promoted direct shoot bud formation from leaf segment explants. In addition, three treatments (4.52 M 2,4-D, 22.71 M TDZ, 4.52 M 2,4-D plus 4.54 M TDZ) gave a higher response than control on mean numbers of shoots per explant with intact leaf explants. Healthy plantlets each with one to three roots were obtained from leaf-derived shoots after transfer onto a hormone-free medium for 22 months. These plantlets were acclimatized in a greenhouse and grew well with 100% survival rate.     

In vitro regeneration of Hagenia abyssinica (Bruce) J.F. Gmel. (Rosaceae) from leaf explants

A protocol for shoot regeneration of Hagenia abyssinica (Bruce) J.F. Gmel. has been developed using leaf explants originating from in vitro seedlings and mature material. The explants were cultured on Murashige and Skoog medium containing various concentrations of -naphthaleneacetic acid and thidiazuron (TDZ). Concentrations of TDZ lower than 1.0 M promoted direct shoot regeneration, but higher concentrations promoted callus induction. Around 96–100% regeneration was obtained between 1.0 and 10 M TDZ. The average number of shoots per explant at 1.0 M TDZ was 8.4±4.8. Among the different explants used, the highest percentage of regeneration and shoots per explant was obtained from complete leaf explants. A significant (P0.05) difference in regeneration capacity was observed among the five genotypes examined. The resulting shoots were multiplied on multiplication medium, rooted and acclimatised in a greenhouse.     

In vitro regeneration of silktree (Albizzia julibrissin) from excised roots

Root segments (1 cm long) were excised from 15–20 day old seedlings of silktree (Albizzia julibrissin) grown on B5 medium. About 50% of the control (no growth regulators added) root explants formed shoot buds within 15 days after placement on the culture medium. After 30 days, there were about 4 shoots per control explant. Addition of low levels of various auxins (0.5 M) did not influence the formation of shoot buds from the explants. Higher concentrations (5M), however, decreased shoot regeneration. Kinetin and 2iP did not influence shoot regeneration at the concentrations tested (1 & 10 M). Addition of benzyladenine, Zeatin, or thidiazuron to the culture medium increased both the percentage of explants that formed shoots and the number of shoots per explant. Thidiazuron was highly effective in stimulating shoot formation at low concentrations (<1 M). At 0.05 M thidiazuron, 95% of the explants produced shoots and about 10 shoots were formed per explant. Compared to TDZ, higher concentrations (10 M) of benzyladenine and Zeatin were required to enhance shoot formation. Upon excision and transfer to B5 medium, regenerated shoots developed into normal rooted plantlets.Abbreviations BA Benzyladenine - IAA Indoleacetic acid - IBA Indolebutyric acid - NAA Naphthaleneacetic acid - TDZ Thidiazuron - 2ip Isopentenyladenine     

Adventitious shoot regeneration from cultured petal explants of carnation

Adventitious shoot regeneration was compared among leaf, stem and petal explants of carnation (Dianthus caryophyllus L.) cv. Scania on MS medium containing different concentrations of 6-benzyladenine (BA) and -naphthaleneacetic acid (NAA). High frequency regeneration was obtained only from petal explants on the media containing 5 to 10 M BA with or without 5 M NAA. Among the cytokinins tested, N-2-chloro-4-pyridyl-N-phenylurea and N-1,2,3-thiadiazol-5-yl-N-N-phenylurea were more effective than BA, kinetin, N⁶-2-isopentenyl adenine and zeatin on regeneration from petal explants. Although, high frequency shoot regeneration was obtained from all petal explants harvested from various developmental stages of buds, a significant decrease in regeneration capacity was observed in the explants obtained from fully-opened flowers. High frequency shoot regeneration was also obtained from the petal explants of cvs. Coral. Lena, Nora and White Sim, and an interspecific cultivar Eolo using the method developed in this study.Abbreviations NAA -naphthaleneacetic acid - BA 6-benzyladenine - GA₃ gibberellic acid - 2iP N⁶-2-isopentenyl adenine - KT-30 N-2-chloro-4-pyridyl-N-phenylurea (also called 4PU) - TDZ N-1,2,3-thiadiazol-5-yl-N-phenylurea (also called thidiazuron)     

In vitro culture ofXanthium strumarium (Cocklebur)

Xanthium strumarium L. was micropropagated by rooting shoots proliferated from shoot-tip explants. The best shoot proliferation was obtained from explants growing on Murashige and Skoog medium supplemented with 4.4 to 8.9 M benzyladenine (BA) and 1.1 to 2.1 M naphthaleneacetic acid (NAA). The micropropagated plants were transferred to potting media and maintained under high humidity conditions in the greenhouse. The media that produced best shoot proliferation from shoot-tip explants also produced the most callus from hypotocotyl, cotyledon and shoot-tip explants, whereas more callus was produced on leaf explants with a lower BA concentration (1.1 M) and 1.1 M NAA.Abbreviations BA benzyladenine, 2 4-d-2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA naphthaleneacetic acid - MS Murashige and Skoog Technical contribution No. 3319 of the South Carolina Agricultural Experiment Station, Clemson University.     

Shoot,root and flower morphogenesis on tomato inflorescence explants

Regeneration of de novo shoots, roots and flowers has been obtained on inflorescence explants of tomato (Lycopersicon esculentum Mill.). Indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and -naphthaleneacetic acid (NAA) were added in a 3×3×3 factorial combination with kinetin, each at 0.001, 0.1 and 10 M concentrations. Direct shoot formation occurred on media with 10 M kinetin and 0.001 M IAA or NAA. Root formation was observed on media with 0.1–10 M IAA, IBA or NAA. Flower formation occurred on elongated shoots with several leaves on media with 10 M IAA and 0.1 M kinetin. Shoot organogenesis was increased by substituting 10 M zeatin or N⁶-benzyladenine (BA) for kinetin. Eleven tomato cultivars were tested for their ability to undergo de novo shoot regeneration on the improved medium. All tomato cultivars were capable of shoot morphogenesis with a mean number of shoots per explant that ranged from 1.3 (Red Alert) to 5.3 (Large Red Cherry). Histological studies revealed that active cell divisions occurred in subepidermal and cambial tisue during the first week of culture. Meristematic centers of dividing cells were evident by day 14, and well-developed shoot apices and leaf structures were observed on 50% of the explants 28 days after culture initiation.Abbreviations BA N⁶-benzyladenine - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - 2iP N⁶-[²-isopentyl]adenine - NAA -naphthaleneacetic acid - PGR plant growth regulator     

Enhancement of shoot regeneration from cotyledon explants of Brassica rapa ssp oleifera through pretreatment with auxin and cytokinin and use of ethylene inhibitors

The presence of benzyladenine or naphthaleneacetic acid in seed germination medium markedly enhanced subsequent shoot regeneration from the base of the excised cotyledon explants of Brassica rapa cv. Horizon. Cotyledon explants from younger seedlings (3 or 4-day old) produced more shoots than those from older seedlings. Addition of the ethylene inhibitor aminoethoxyvinylglycine (1.0 M) to the regeneration medium improved shoot regeneration three fold.Abbreviations AVG aminoethoxyvinylglycine - BA benzyladenine - MGBG methylglyoxal-bisguanylhydrazone - MSBN ms (murashige & skoog 1962) medium supplemented with 4.4 m BA & 5.4 m NAA, 2% sucrose - NAA naphthaleneacetic acid     

Organogenesis and somatic embryogenesis in callus cultures ofRosa hybrida andRosa chinensis minima

Pre-incubation of somatic tissues of the cut rose Carefree Beauty and miniature roses Red Sunblaze and Baby Katie in 10, 100, or 200 M 2,4-D induced the development of highly rhizogenic callus. Upon transfer of rhizogenic callus to a regeneration medium containing 23 M TDZ and 3 M GA₃, a low frequency of shoot organogenesis (3.3%) and somatic embryogenesis (6.6%) was observed. Incubation of leaf and stem internodes in 11, 27, 54, 81, or 108 M NAA followed by transfer of explants to the regeneration medium resulted in up to a 25% increase in shoot organogenesis from callus-derived internodal explants of Red Sunblaze, but no somatic embryogenesis was observed. The influence of glucose versus sucrose in the regeneration medium on leaf explants of Carefree Beauty and Baby Katie pre-incubated in 100 M 2,4-D revealed a difference in genotypic response to shoot organogenesis and somatic embryogenesis. For Carefree Beauty, a concentration of 111 mM glucose induced higher frequencies of both organogenic (33%) and embryogenic calluses (25%) than either 59 mM or 117 mM sucrose. For Baby Katie, no significant difference was found for number of organogenic calluses induced on 59 mM sucrose and 111 mM glucose.Abbreviations ABA Abscisic acid - BA Benzyladenine - 2,4-D 2,4-Dichlorophenoxyacetic acid - GA₃ Gibberellic acid - IAA Indoleacetic acid - MS Murashige & Skoog (1962) - NAA -Naphthaleneacetic acid - TDZ Thidiazuron     

Regeneration of Elaeagnus angustifolia from leaf segments of in vitro-derived shoots

Shoot regeneration was achieved from in vitro-produced leaves of Elaeagnus angustifolia L. Half-leaf explants from the terminal part of the shoot produced more shoots than explants from the basal part of the in vitro-derived shoots on agar-solidified WPM medium supplemented with 1 M benzyladenine (BA). In liquid medium of the same formulation, compact shoots that did not elongate were formed on the explants. Leaf cross-section explants (1 mm thick) produced shoots both on solid and liquid medium with 1 M BA, whereas again compact shoots were formed with 10 M BA. Further shoot development on these explants was promoted by their transfer to fresh solid medium containing 1 M BA and 1 M gibberellic acid (GA₃).Abbreviations BA benzyladenine - GA₃ gibberellic acid - WPM woody plant medium     

In vitro regeneration of chickpea (Cicer arietinum L.): Stimulation of direct organogenesis and somatic embryogenesis by thidiazuron

In vitro regeneration in chickpea (Cicer arietinum L.) was achieved by direct culture of mature seeds on Murashige and Skoog (MS) medium supplemented with either N-phenyl-N(-1,2,3-thidiazol-5-yl) urea (thidiazuron, TDZ) or N⁶-benzylaminopurine (BAP). Multiple shoots formed de novo without an intermediary callus phase at the cotyledonary notch region of the seedlings within 2 to 3 weeks of culture initiation. TDZ was found to be more effective compared to BAP as an inductive signal of regeneration. The former induced multiple shoot formation at all the concentrations tested (1 M to 100 M), although, maximum morphogenic response was observed at 10 M concentration. Addition of naphthaleneacetic acid (NAA) alone or in combination with BAP to the MS medium failed to invoke a similar response. When the TDZ supplemented medium was amended with L-proline, the resultant regenerants were mostly somatic embryos. Histological investigations confirmed the switch in the regeneration pathway from directly formed adventitious shoots to embryogenesis. For obtaining plantlets, adventitious shoots were rooted on MS medium supplemented with 2.5 M NAA; somatic embryos were germinated and established on MS medium. Normal plants were regenerated from both adventitious shoots and somatic embryos and transferred to soil.Abbreviations BAP 6-benzylaminopurine - MS Murashige and Skoog [14] basal medium - NAA naphthaleneacetic acid - TDZ thidiazuron [N-phenyl-N(-1,2,3,-thidiazol-5-yl)-urea]     

In vitro regeneration through organogenesis of Meconopsis simplicifolia — An endangered ornamental species

Meconopsis simplicifolia (D.Don) Walp. could be propagated by induction of adventitious shoots from callus produced on hypocotyl, cotyledon and rosette leaf explants of 4-month-old seedlings. Callus was initiated on agar-solidified Murashige and Skoog medium supplemented with 1 M kinetin +10 M -naphthaleneacetic acid (NAA). Shoots formed when the callus was subcultured on medium supplemented with kinetin or benzyladenine (BA) in combination with NAA, indoleacetic acid, indolebutyric acid or gibberellic acid. Excised shoots were rooted on medium containing auxin with 10 M NAA producing the best rooting (55%).Abbreviations BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxy-acetic acid - FAA formalin-acetic acid-alcohol - GA₃ gibberellic acid - IAA indole-3-acetic acid - IBA -indolebutyric acid - NAA -naphthaleneacetic acid     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of the medicinal woody plant <Emphasis Type="Italic">Phellodendron amurense</Emphasis> Rupr. through excised leaves

Shoot organogenesis and plant establishment has been achieved for Phellodendron amurense Rupr. from excised leaf explants. Young leaf explants were collected from in vitro established shoot cultures and used for the induction of direct shoot regeneration, callus and subsequent differentiation into shoots on MS medium. Direct shoot regeneration was achieved by culturing 1 cm² sections of about 10-day-old leaves on MS medium enriched with 4.4 M BAP and 1.0 M NAA after 4 weeks of culture. The leaf explants produced callus from their cut margins within 3 weeks of incubation on medium supplemented with 2.0 M TDZ and 4.0 M 2,4-D or 4.0 M NAA. The maximum number of adventitious shoots was regenerated from the leaf-derived callus within 4 weeks of culture on MS medium containing 1.5 M BAP and 1.0 M NAA. The highest rate of shoot multiplication was achieved at the third subculture, and more than 65 shoots were produced per callus clump. For rooting, the in vitro proliferated and elongated shoots were excised into 2–4 cm long microcuttings, which were planted individually on a root-induction MS medium containing 2.0 M IBA. Within 3 weeks of transfer to the rooting medium, all the cultured microcuttings produced 2–6 roots. The in vitro regenerated plantlets were transferred to Kanuma soil, and the survival rate ex vitro was 90%.     

Adventitious shoot regeneration from leaf explants of tissue cultured and greenhouse-grown raspberry

Adventitious shoot regeneration was observed using leaf-petiole explants from shoot-proliferating cultures of Comet red raspberry (Rubus idaeus L.). A maximum regeneration rate of 70% (3.7 shoots/explant) was obtained using 4.5–9.1 M (1–2 mg l^–1) N-phenyl-N-1,2,3-thiadiazol-5-ylurea (thidiazuron or TDZ) with 2.5–4.9 M (0.5–1 mg l^–1) 1H-indole-3-butanoic acid (IBA) or 2.3 M (0.5 mg l^–1) TDZ with 4.9 M (1 mg l^–1) IBA in modified Murashige-Skoog medium. TDZ was more effective than N-(phenylmethyl)-1H-purin-6-amine (BA) at promoting regeneration in combinations tested with IBA (maximum 50% regeneration rate; 1.8 shoots/explant). Variation in the agar concentration or incubation temperature, orientation or scoring of the leaf-petiole explants and use of separate leaf or petiole explants had no effect on shoot regeneration. Incubation in the dark for 1, 2 or 3 weeks prior to growth in the light did not influence the percent regeneration rate but depressed the number of adventitious shoots. Explant source, from micropropagated shoots or greenhouse-grown plants, had an effect on shoot regeneration that was genotype dependent. Only 8 of 22 (36%) raspberry cultivars were capable of regeneration from leaf explants derived from greenhouse-grown plants.     

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     

Factors influencing shoot regeneration from cotyledonary explants ofCucumis melo

Cotyledonary explants of 4-day-oldCucumis melo cv. Hale's Best Jumbo in vitro seedlings showed maximum initiation of shoot buds when cultured onto a revised Murashige & Skoog medium supplemented with 5 M indole-3-acetic acid and 5 M benzylaminopurine and cultured at 25–29°C under low light intensity (5–30 mol m^-2 s^-1). Subculture of the shoot buds onto the same medium without auxin and supplemented with 3 M benzylaminopurine caused the development of shoots from 30% of the buds. The presence of abscisic acid significantly increased the number of explants producing shoot buds. Bud initiation was affected by genotype, seedling age, light intensity, and temperature. Addition of gibberellic acid, thidiazuron or silver nitrate to regeneration medium did not improve either bud initiation or shoot regeneration.     

In vitro meristem cloning of Eucalyptus tereticornis Sm.

Rapid multiplication of axillary meristems and direct shoot development occurred from nodal explants of mature Eucalyptus tereticornis Sm. with 5.3 M NAA, 1.1 M IAA and 4.4 M BA in Murashige-Skoog medium. Repeated subcultures of the second generation shoot cultures into low cytokinin-auxin containing media (0.44–0.88 M BA+0.1 M NAA) yielded axillary microshoots in large numbers. Half-strength MS liquid medium with 4.9 M IBA, 5.5 M IAA and 5.3 M NAA for four days, half-strength semi-solid hormonefree MS medium with charcoal, and MS liquid medium without charcoal and hormones, in sequence, induced rooting of shoots in the dark. This system is suitable for the mass propagation of this difficult-to-root eucalypt.Abbreviations BA 6-benzyladenine - IAA indole-3-acetic acid - IBA -indole-3-butyric acid - 2-iP isopentyl adenine - Kn kinetin - MS Murashige-Skoog - NAA -naphthalene acetic acid - PVP polyvinylpyrrolidone     

In vitro shoot regeneration from leaf explants of Roman Chamomile

Murashige and Skoog (1962) medium supplemented with 1.0 to 4.5 M of BA and 1.0 M of NAA induced adventitious bud formation and shoot development in leaf explants of Roman Chamomile. A higher number of adventitious buds was observed at the proximal end of the explants. Plantlets were replicated and multiplied on MS medium supplemented with 2.25 M of BA and 0.6 M of IAA. Plantlets were rooted on MS medium supplemented with 0.5 M of IBA and successfully weaned in vivo. The plants grew to maturity with high uniformity and no morphological signs of somaclonal variation.     

In vitro clonal propagation of Pisum sativum L.

A system of in vitro clonal propagation has been developed in Pisum sativum L. (cv. Bohatýr). A modified MS-medium supplemented with 20 M 6-benzylaminopurine (BAP) and 0.1 M -naphthaleneacetic acid (NAA) was used to induce multiple shoot formation from shoot apices, axillary buds of the first normal leaf, axillary buds of the first and second primary scales and axillary buds of cotyledons of 4 to 6 day old pea seedlings. Meristem explants maintained a high proliferation ability in each subculture in the course of 20 months of the culture. Regenerated shoots were rooted in the same basal medium containing 5 M NAA. Rooted plants were cultured in hydroponic pots filled with half-strength MS-medium to attain anthesis and seed maturity. The phenotypic uniformity of the regenerants was evaluated. Cytological investigation confirmed the diploid stage (2n=14) of regenerants and their progeny. Histological studies revealed that proliferating shoots originated from axillary and adventitious buds. In vitro propagation is discussed as related to pea breeding.     

Plant regeneration from seedling explants of Eucalyptus grandis × E. urophylla

Hypocotyls, cotyledons, cotyledonary nodes and primary leaves were used as explants to establish a regeneration protocol for Eucalyptus grandis × E. urophylla. These seedling-derived explants were incubated on a modified MS medium (SP medium), supplemented with 2.0 M TDZ. After 1 month, the calluses obtained were transferred to SP medium containing different concentrations of BA and NAA or zeatin and NAA. Shoots were induced from these calluses at a high frequency. Shoot elongation was then stimulated on SP medium supplemented with BA, NAA and GA₃ for 20 to 30 days. For rooting, 50 mm long shoots were cultivated on root induction medium containing IBA (2.5 M) for different periods and then transferred to the same medium but without auxin, for 30 days. Plantlets were then successfully transplanted to greenhouse conditions.     

Effect of genotype,explant and medium onin vitro regeneration of red pepper

In vitro plant regeneration was achieved inCapsicum praetermissum, C. baccatum andC. annuum cvs. G4, Bhiwapuri Sweet pepper, Cayenne pepper and Hybrid pepper. Shoots were induced from hypocotyl, cotyledon and leaf explants on Murashige and Skoog medium supplemented with 5.7 M indoleacetic acid (IAA)+13.3 M benzyladenine (BA); 22 M BA; and 44 M BA. Analysis of variance revealed that the most significant effect on shoot regeneration was due to the explant and it accounted for 56.3% of total variation observed. The genotype x explant effect on regeneration was minor relative to all other 2- and 3-way interactions because leaf explants consistently regenerated more shoots than hypocotyls or cotyledons in all the genotypes and thereby reduced the variation among the genotypes. Explant x medium interaction revealed that 22 M BA was the best growth regulator supplement in regeneration medium for optimal shoot regeneration from leaf explants. Rooting of regenerated shoots was achieved on 5.7 M IAA-containing medium, and the rooting response was better from shoots induced on medium fortified with 5.7 M IAA plus 13.3 M BA. Complete plantlets with diploid chromosome number (2n=2x=24) were transferred to soil and 60–70% of these plantlets survived and grew well.