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     

Plant regeneration from callus cultures derived from mature zygotic embryos in white pine (Pinus strobus L.)

Plant regeneration via adventitious shoot organogenesis from callus cultures initiated from mature embryos in white pine (Pinus strobus L.) was achieved in this study. Callus cultures were induced from mature embryos cultured on PS medium supplemented with 2,4-dichlorophenoxyacetic acid, -naphthaleneacetic acid, or indole-3-acetic acid. Adventitious shoot regeneration from callus cultures was induced on medium containing 2 M indole-3-butyric acid (IBA) and 3–12 M N₆-benzylaminopurine, thidiazuron (TDZ), or 6-(,-dimethylallylamino) purine. Sucrose was the most suitable sugar for adventitious shoot organogenesis in white pine. Shoot organogenesis was improved by treatment at 4°C for 6 weeks. The frequency of adventitious shoot formation increased when 0.1 mM putrescine was added to basal medium supplemented with 6 M TDZ and 2 M IBA. Putrescine improved adventitious shoot organogenesis by decreasing lipid peroxidation. These findings provide useful information on adventitious shoot organogenesis and may be valuable to genetic transformation in white pine.     

An efficient adventitious shoot regeneration system for Zhanhua winter jujube (Zizyphus jujuba Mill.) using leaf explants

The direct induction of adventitious shoots from leaf explants obtained from adult plants of Zhanhua winter jujube, an elite variety of Zizyphus jujuba Mill., is reported. The percentage of leaf explants producing shoots and the average number of shoots per explant were significantly improved when 10-day-old leaves were explanted onto Woody Plant Medium and maintained initially in the dark. The plant growth regulator thidiazuron (TDZ) was effective in stimulating shoot regeneration from leaf explants of Zhanhua winter jujube. The highest efficiency of shoot formation was observed with a 20-day culture in the dark on WPM containing 4.54 M TDZ and 2.85 M indoleacetic acid (IAA). The regenerated shoots were transferred to MS medium containing 0.89 M benzyladenine and 5.77 M gibberellic acid for growth. When the shoots were about 2 cm in height, they were transferred to Nitsch medium supplemented with 1.14 M IAA and 2.46 M indolebutyric acid to induce rooting. This system of adventitious shoot production from leaf explants of adult plants could be useful for the genetic engineering and polyploidization of winter jujube.     

In vitro shoot regeneration from olive cultivar tissues

Petioles, leaf discs and midribs of several olive (Olea europaea L.) cultivars, collected from potted greenhouse plants, field-grown and in vitro shoots, were used to test their morphogenic capacity. Adventitious shoots were induced only in petioles from in vitro-grown shoots of cultivars Moraiolo, Dolce Agogia and Halkidikis, grown on Olive Medium (OM) plus 18 M zeatin within 4 to 5 weeks. Regeneration was achieved, both on Murashige and Skoog (MS) and on modified OM, only in the dark. The highest regeneration was achieved directly from the proximal part of the petioles after 2 to 3 weeks in media containing 5 to 40 M thidiazuron, or with both 10 M 2-isopentenyladenine +2.2 M 6-benzyladenine with or without low auxin concentration (not more than 2.5 M). A few adventitious shoots were also regenerated from callus when it was shifted from auxin and cytokinin media to cytokinin only medium. The regeneration potential was higher in petioles collected from apical nodes than from basal ones. The adventitious shoots were transferred to solid half-strength MS medium supplemented with 4.5 M zeatin for further development. Several regenerated shoots were rooted and the plantlets hardened in the greenhouse. No apparent differences regarding morphological aspects were observed among the regenerated plantlets or with those obtained by stimulation of axillary buds.Abbreviations BA 6-benzyladenine - IBA indole-3-butyric acid - NAA 1-naphthaleneacetic acid - TDZ thidiazuron (N-phenyl-N-1,2,3-thidiazol-5-ylurea) - 2iP 2-isopentenyladenine - MS Murashige and Skoog medium - 1/2 MS half strength MS - OM Olive Medium - BN Bourgin & Nitsch     

Rapid mass propagation of Tylophora indica Merrill via leaf callus culture

An efficient protocol has been developed for rapid mass propagation of Tylophora indica from leaf derived callus. Optimal callus was developed from leaf explants on Murashige and Skoog (MS) basal medium supplemented with 10 2,4,5-T. Adventitious shoots were regenerated (85%) from the surface of the callus on MS medium supplemented with 5 M Kinetin. Individual elongated shoots were rooted on half-strength MS medium containing 0.5 M IBA. Regenerated plantlets with well developed shoots and roots were successfully transferred to soil. The study demonstrated a dedifferentiated callogenic propagation route via adventitious shoot development in T. indica, which could be useful for large scale multiplication of this endangered medicinal plant.     

Development of high frequency multiple shoot formation in Persian clover (<Emphasis Type="Italic">Trifolium resupinatum</Emphasis> L.)

An efficient and reliable micropropagation system for Persian clover (Trifolium resupinatum L.) was developed using different explants and media. Node, hypocotyl and cotyledonary node explants were cultured on Murashige and Skoog (MS) medium supplemented with combinations of either 6-benzyladenine (BA) and indole-3-butyric acid (IBA) or BA, Kinetin (KIN) and IBA. Direct multiple shoots developed within 6weeks in all explants in most media tested. The best shoot multiplication capacity was obtained from cotyledonary node explants on MS medium containing 7.1M BA and 1M IBA or 14.1M BA and 1M IBA. Elongated shoots were rooted on either MS medium alone or combination with different concentrations of indole-3-butyric acid (IBA), indole-3-acetic acid (IAA) and -naphthaleneacetic acid (NAA). High rooting was achieved in half strength MS medium containing 8M IBA.     

Clonal Propagation of Dwarf Raspberry (Rubus pubescens Raf.) through in vitro Axillary Shoot Proliferation

A protocol for the micropropagation of dwarf raspberry (Rubus pubescens) was developed by the establishment of axenic shoot cultures from greenhouse-grown plants, induction of shoot proliferation, and rooting in vitro. Cultures were initiated from shoot tip and nodal explants on 1/2 strength MS (Murashige T. and Skoog F. 1962. Physiol. Plant. 15:473) macro-salts and MS micro-salts and vitamins containing 8.9 M N ⁶-benzyladenine (BA) and 0.98 M indole-3-butyric acid (IBA). Zeatin was more effective than BA, and induced proliferation of about 1.5–2 times as many shoots as BA in combination with 0.54–1.1 M -naphthaleneacetic acid (NAA) or 0.49–0.98 M IBA. With higher zeatin, shoots did not expand and had a high mortality rate. Shoots growing for more than 10 weeks on medium that contained 9.1 M zeatin occasionally produced adventitious shoot masses, which appeared to arise from dense calluses growing at the base of the shoots in the medium. Shoots were rooted in vitro in the same medium used for shoot proliferation, but without any growth regulators. Almost all (85–90%)in vitro plantlets survived when transferred to potting medium.     

Adventitious regeneration of Juglans nigra L. (eastern black walnut)

Somatic embryos and adventitious shoots were initiated from immature cotyledons 10–14 weeks after anthesis. Maximum embryogenesis occurred 12 weeks after anthesis and maximum shoot organogenesis occurred 14 weeks after anthesis. The best treatment for induction of somatic embryos and adventitious shoots from immature cotyledon explants was on agar-solidified WPM supplemented with 0.1 M 2,4-D and 5.0 M TDZ and incubated in light for the first four weeks. Rooting of adventitious shoots was best if they were quickdipped in 2.5 mM IBA and 1.25 mM NAA in 1% dimethyl formamide and 3.9% ethanol (120 Wood's Rooting Compound: water, by volume). Plantlets from rooted adventitious shoots were acclimatized to the greenhouse.Abbreviations BA Benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - DKW Driver and Kuniyuki (1984) walnut medium - IBA indolebutyric acid - LP Long and Preece medium described herein - NAA naphthaleneacetic acid - PPF photosynthetic photon flux - TDZ thidiazuron - WPM Woody Plant Medium of Lloyd and McCown (1980)     

In vitro organogenesis and plant formation in Vigna radiata (L.) Wilczek

In vitro organogenesis was achieved from calluses derived from cotyledon and hypocotyl explants of Vigna radiata on MS medium. Organogenic calluses were induced from both cotyledon and hypocotyl explants excised from 3-day-old seedlings on MS medium containing NAA (1.07 m and BA (2.22 m) and 2,4-D (0.90 m) and BA (2.22 m) combinations respectively. Regeneration of adventitious shoots from cotyledon derived callus was achieved when they were cultured on MS medium supplemented with NAA (1.07 m), BA (8.88 m) and 10% coconut water. Hypocotyl derived calluses produced adventitious shoots when cultured on MS medium fortified with BA (6.66 m), TDZ (2.5 m) and 10% coconut water. Addition of GA at 1.73 m favored maximum 3 elongation of shoots. Regenerated shoots produced prominent roots when transferred to half strength MS medium supplemented with 4.90 m IBA. Rooted plantlets, thus developed were hardened and successfully established in field. Among the different carbohydrates and media tested, 87.64 m sucrose and MS+B5 medium proved best for maximum production of shoots. This protocol produced an average of seven plants per hypocotyl derived callus and 15 plants per cotyledon derived callus over a period of 3 months.     

Indole butyric acid induces regeneration of phenotypically normal apricot (Prunus armeniaca L.) plants from immature embryos

A prerequisite for most transformation systems is an efficient and reliable method to regenerate phenotypically normal plants. Immature embryos or cotyledons were cultured at three developmental stages (stage 1, 2 and 3, PF=3, 30–60, and 100, respectively) from two unrelated apricot genotypes, Zard and NJA82. Explants were cultured on MS media supplemented with either BA or TDZ at four concentrations (0, 0.5, 5.0 or 20 M) and 2,4-D at 0 or 1 M. Stage 1 embryos cultured on MS medium without growth regulators formed embryoid-like structures. Shoot primordia induction was greatest with stage 2 cotyledons on media containing 5–20 M TDZ and 1 M 2,4-D, although shoot morphology was abnormal, especially with the highest level of TDZ. In another factorial experiment, stage 2 cotyledons were cultured on media containing TDZ (0, 5, 7.5, 10, 15 or 20 M) in combination with either no auxin, 1 M 2,4-D, 1 M IBA, or 5 MIBA. Regeneration percentages of 80% or more were observed on media containing 1–5 M IBA and 5–10 M TDZ. The medium containing 5 M IBA and no TDZ exhibited the highest frequency of phenotypically normal plantlet regeneration.Abbreviations BA 6-benzylaminopurine - IBA indole-3-butyric acid - 2,4-D 2,4 dichlorophenoxyacetic acid - 2iP 2-isopentenyladenine - MS Murashige and Skoog basal medium - NAA 1-naphthaleneacetic acid - PF percent fill [(embryo length/seed length) × 100] - TDZ thidiazuron [N-phenyl-N(1,2,3,-thidiazol-5-yl)-urea] - WPM McCown's woody plant medium     

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.     

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     

An in vitro microplant bioassay using clonal white ash to test for tall fescue allelopathy

Axillary shoots from three selected white ash (Fraxinus americana L.) clones were harvested from in vitro shoot cultures. Roots were initiated by pulsing excised shoots for eight days in the dark in MS medium supplemented with 2% sucrose, 0.7% agar, 5 M NAA, and 1 M IBA. Pulsed shoots were transferred to a root elongation medium consisting of 25% MS macrosalts, full-strength microsalts and organics, 1% sucrose, 0.7% agar and no auxins. When roots were visible (6–10 days after transfer to root elongation medium), microplants were transferred to vessels containing the same minimal medium and tall fescue (Festuca elatior var. arundinacea (Schreb.) Wimm.) leaf extracts, leaf leachates, or soil leachates from plant boxes with and without tall fescue sod. After four weeks in vitro, primary adventitious and secondary root growth was reduced by extracts obtained from 5 and 10 g ground leaves per 100 ml of medium. Leachates obtained from 5 g soaked leaves per 100 ml of medium stimulated primary root growth. Soil leachates from bare soil also stimulated primary root growth. Variation was observed among the clones for root growth when plantlets were grown in extracts or leachates from tall fescue.     

Rapid clonal propagation of Dioscorea zingiberensis

A protocol was developed for rapid in vitro propagation of Dioscorea zingiberensis Wright using stems as explants. MS medium with the macroelements at half strength and supplemented with 20.0 g l^–1 sucrose and 8.0 g l^–1 agar was used as basal medium. Lateral buds on nodal cuttings grew into shoots within 20 days after culture on basal medium supplemented with 4.4 M 6-benzylaminopurine (BAP) and 1.1 M -naphthalene acetic acid (NAA). The shoots were cut into segments and cultured on medium with 8.9 M BA and 5.4 M NAA for 30 days for callus formation. The callus was cut into pieces and cultured on medium containing 22.2 M BAP and 1.1 M NAA, on which 87.5% of the callus pieces regenerated multiple shoots within 50 days. The shoots were rooted on medium containing 4.9 M indole-3-butyric acid (IBA) for 20 days. Adventitious buds and shoots could be repeatedly formed after the calli were cut into pieces and cultured on the medium containing 8.9 M BAP plus 1.1 M NAA. More than 85% of the regenerated plantlets survived and grew vigorously 1 month after they were transplanted in vermiculite and each plant formed 2–4 microtubers 3 months of transplanting.     

Shoot multiplication in <Emphasis Type="Italic">Rauvolfia tetraphylla</Emphasis> L. using thidiazuron

The effect of thidiazuron (TDZ) was investigated on in vitro shoot proliferation from nodal explants of Rauvolfia tetraphylla. Murashige and Skoog (MS) medium containing TDZ (0.5–10M) was effective in inducing shoot buds and maintaining high rates of shoot multiplication on hormone free medium. The highest shoot regeneration frequency (90%) and mean number (18.50 ± 1.25) of shoots per explant were achieved from nodal segments cultured on MS medium supplemented with 5M TDZ for 4 weeks prior to transfer to MS medium without TDZ for 8 weeks. The regenerated shoots rooted best on MS medium containing 0.5M indole-3-butyric acid (IBA). Micropropagated plantlets were hardened to survive ex vitro conditions and were then established into soil.     

Effects of thidiazuron and N6-benzylaminopurine on shoot regeneration of Phalaenopsis

Adventitious bud formation from the vegetative buds of the flower stalks of Phalaenopsis occurred on Vacin and Went medium with 15% coconut water and 5 to 40 M thidiazuron (TDZ) or 40 M N⁶-benzylaminopurine. The highest efficiency of induction was achieved with 5 or 10 M TDZ. Adventitious buds developed into shoots on VWC medium. TDZ was more effective than BAP in stimulating the axillary buds of intact shoots to develop. Regenerated shoots rooted after about two months of culture on VWC medium with 1% sucrose. Shoot tips excised from the regenerated shoots initiated protocorm-like bodies after two months of culture on VWC medium.Abbreviations VWC medium Vacin and Went medium with 15% (by volume) coconut water - TDZ thidiazuron - BAP N⁶-benzylaminopurine - Plbs protocorm-like bodies     

Adventitious shoot regeneration from leaf explants of almond (Prunus dulcis mill.)

Summary A method has been developed to facilitate shoot formation from leaf explants of almond. Leaves were dissected from micropropagated shoot cultures of the commercial cultivars Nonpareil and Ne Plus Ultra, and sections incubated on Almehdi and Parfitt's (1986) basal medium (AP) with varied plant growth-regulator conditions. Three auxins, 2,4-dichlorophenoxyacetic acid (2,4-D), α-naphthaleneacetic acid (NAA), and indole-3-butyric acid (IBA), in combination with two cytokinins, benzylaminopurine (BA) and thidiazuron (TDZ), were tested at various concentrations along with casein hydrolysate (CH) to determine, the conditions most conducive to adventitious shoot regeneration. Response to the tested plant growth-regulator conditions varied with genotype. Of the three auxins tested, NAA and IBA induced adventitious shoots from Ne Plus Ultra explants, but only IBA was effective for Nonpareil. For the cytokinins, shoot development from Ne Plus Ultra occurred in the presence of either BA or TDZ, whereas for Nonpareil only TDZ was effective unless CH was incorporated in the basal medium. The inclusion of CH (0.1% w/v) improved callus morphology, and increased regeneration frequencies for both cultivars. Maximum regeneration frequencies for Ne Plus Ultra (44.4%) and Nonpareil (5.5%) were achieved on AP basal salts supplemented with CH, IBA (9.8 μM), and TDZ at 22.7 and 6.8 μM, respectively.     

TDZ-induced somatic embryogenesis in non-responsive caryopses of rice using a short treatment with 2,4-D

Caryopses cultures of rice on an auxin medium (2,4-D, 20 M) formed slow-growing tissues that failed to regenerate. The embryogenic tissue possibly lost its regeneration potential on the auxin medium. Reproducible regeneration, however, could be achieved by a short treatment with 20 M 2,4-D for 3 days. Transfer of these caryopses to the medium containing TDZ or BA at 10 M resulted in regeneration of somatic embryos and shoots in 30% of the cultures within 10–15 days. TDZ was better than BA for inducing shoot regeneration. Short treatment for 3 days at higher concentrations of auxin (40–80 M) and subsequent transfer to TDZ or BA medium resulted in an increased frequency (up to 50%) of regeneration.     

Micropropagation of Tamarix gallica from nodal explants of mature trees

Tamarix gallica L. was micropropagated from four-to six-node explants taken from mature trees. Shoot proliferation was induced on Linsmaier and Skoog medium containing 30 g l^-1 sucrose, 7 g l^-1 agar, 200 mg l^-1 reduced glutathione (basal medium) and supplemented with 3.3 M benzyladenine. Adding 0.5 or 1.0 M indole-3-butyric acid (IBA) to the basal medium increased lateral shoot formation and ease of rooting. Microcuttings repeatedly subcultured on 1.0 M IBA produced well-developed roots, a high number of axillary shoots and could be acclimatized in the greenhouse.     

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.