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     

Adventitious shoot formation from leaf explants of Rhododendron

A protocol for high frequency adventitious shoot regeneration adventitious shoot regeneration from leaf explants of Rhododendron spp. has been developed. The highest percentage of regeneration and the greatest number of shoots were obtained when leaf explants were cultured on Anderson's medium containing 4.9 M IBA and 73.8 M 2iP. Genotypic variation was observed for adventitious shoot regeneration potential among the seven cultivars tested. Regeneration frequencies ranged from 0 to 96%. Lodestar had the highest rate of regeneration after 3 months of culture with 96% shoot regeneration and an average of 14 shoots per explant. Regenerated shoots were rooted in soil in about 2 months. This protocol should be useful in applying gene transfer techniques to Rhododendron improvement.Abbreviations IAA 1-H-indole-3-acetic acid - NAA 1-naphthaleneacetic acid - IBA 1-H-indole-3-butyric acid - 2,4-d 2,4-dichlorophenoxyacetic acid - BA 6-benzyladenine - 2iP N-(3-methyl-2-butenyl)-1-H-purine-6-amine     

Thidiazuron-induced adventitious shoot regeneration of sweetpotato (Ipomoea batatas)

Adventitious shoots of sweetpotato (Ipomoea batatas L. Lam.) were produced in vitro using a two-stage culture method. Petiole explants were incubated on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxy acetic acid (0.2 mg·liter^−1) for 3 d, and transferred to MS medium with thidiazuron (0 to 0.4 mg·liter^−1). Shoot regeneration was observed in most explants (78.2%) of genotype PI 318846-3 within 28 days when cultured on thidiazuron at 0.2 mg·liter^−1. Histological studies of cultured petiole explants showed meristematic activity within cells of vascular bundles and throughout the ground tissue. Explants isolated from apical leaves exhibited higher shoot regeneration frequency than those isolated from the basal portion of the shoot. Leaf lamina explants exhibited lower frequency of regeneration than petiole explants. In contrast to thidiazuron, the use of zeatin riboside, and kinetin resulted in a lower frequency of shoot regeneration although more sweetpotato genotypes could be regenerated using either of these two cytokinins. The sweetpotato plants regenerated using thidiazuron grew vigorously and rooted easily when transferred to the greenhouse.     

Thidiazuron stimulates adventitious shoot regeneration in different safflower explants

Adventitious shoot regeneration from root, hypocotyl, cotyledon and primary leaf explants of safflower (Carthamus tinctorius L.) was studied. Shoot regeneration was promoted by benzyladenine (BA) + naphthaleneacetic acid (NAA), BA + indole-3-butyric acid (IBA), kinetin + NAA and thidiazuron (TDZ) + NAA incorporated in Murashige and Skoog (MS) basal medium. High frequency of shoot regeneration and high number of shoots per regenerating explant were obtained on a wide range of TDZ + NAA combinations. Proliferated shoots were elongated in MS + 0.5 mg dm⁻³ kinetin and well-developed shoots were rooted in half strength MS + 0.5 mg dm⁻³ NAA. Rooted shoots were successfully acclimatized and established in soil.     

Thidiazuron promotes in vitro regeneration of wheat and barley

Summary Thidiazuron (TDZ) is a substituted phenylurea which has been shown to be an efficacious regulator of in vitro morphogenesis of many dicot plant species. However, information regarding the effect of TDZ on in vitro regeneration of monocot species is limited. We investigated the effects of TDZ on in vitro regeneration of barley (Hordeum vulgare L.) and wheat (Triticum aestivum L.) and found that it promoted shoot regeneration from callus in these two important cereal species. Plant regeneration from calluses derived from immature embryo culture of barley and wheat was observed in regeneration media with a wide range of TDZ concentrations (0.045–45 μM). Shoot regeneration from barley calluses was the highest (38.3% for cv. Golden Promise) at 4.5 μM (1 mg l⁻¹) TDZ, while the optimal TDZ level for wheat regeneration seemed to be 0.9 μM (0.2 mg l⁻¹) (87% for cv. Bob White and 49.4% for cv. Hi Line). Roots developed normally when the regenerated wheat and barley shoots from TDZ-containing media were transferred to the rooting medium. Comparison with other plant growth regulators commonly used in wheat and barley regeneration media suggested that TDZ was among the best for in vitro regeneration of wheat and barley. Both authors contributed equally     

adventitious shoot regeneration from petiole explants of Heracleum candicans wall

Summary A method for adventitious shoot induction from petiole explants of Heracleum candicans is reported. Shoot buds were induced on Murashige and Skoog (MS) medium with 4.4μM 6-benzylaminopurine (BA) and 1.1 μM 2,4-dichlorophen-oxyacetic acid (2,4-D). A wound response in the presence of BA and 2,4-D at the time of culture was necessary for inducing shoot buds. The shoot bud regeneration was significantly influenced by size, type and orientation of explants on the culture medium. These shoot buds developed into 4–5 cm shoots upon transfer to a medium containing 1.1μM BA and 0.5 μM α-naphthaleneacetic acid (NAA). The regenerated shoots formed rooted plantlets on MS medium supplemented with 4.9 μM indole-3-butyric acid (IBA). About 15 plants were established in the field for further evaluation.     

Shoot organogenesis from petiole explants in the aquatic plant Nymphoides indica

A protocol for rapid shoot organogenesis from petiole explants of the ornamental aquatic plantNymphoides indica L. Thwaites O. Kuntze was developed for use in future mutation breeding and cultivar selection studies. Optimum culture conditions for shoot organogenesis were determined. Effects of factorial combinations of 2-iP, BA or kinetin (0–25 μM) in factorial combination with IAA or NAA (0–25 μM) were examined. On the basis of regeneration frequency (80%) and adventitious shoot number (11.5 shoots per explant), most efficient shoot organogenesis occurred on petiole explants cultured on a basal medium consisting of full-strength MS inorganic salts, 0.56 mM myo-inositol, 1.2 μM thiamine-HCl, 116.8 mM sucrose supplemented with 10 μM BA and 20 μM IAA and solidified with 0.8% TC agar. Formation of adventitious shoots by direct and indirect shoot organogenesis from the same explant was verified by histological sectioning. With the exception of variegated leaf production on a single adventitious shoot produced in the presence of 25 μM kinetin and 15 μM NAA, no visible phenotypic abnormalities were observedin vitro in any of the shoots generated. Solid achlorophyllous adventitious shoots were recovered following culture of this variegated leaf tissue. Plantlets were easily acclimatized toex vitro conditions. This revised version was published online in June 2006 with corrections to the Cover Date.     

Influence of petiole and lamina on adventitious shoot initiation from leaf explants of Paulownia fortunei

Adventitious shoot bud differentiation occurred preferentially from the petiolar cut ends of leaf explants of Paulownia fortunei cultured on Murashige and Skoog medium supplemented with 4 μmα-naphthaleneacetic acid and 20 μm benzyladenine. The details of plantlet regeneration and successful transplantation to soil have been reported earlier. We now show that besides medium supplementation with auxin and cytokinin, the presence of lamina and petiole in the explant influence shoot bud induction. Explants with the basal half of the lamina and the entire petiole were much more responsive than those with whole lamina and petiole. A dual-culture-medium technique which permitted incubation of the two ends of excised petioles under two different phytohormone regimes was devised. Our data suggest that some of the diffusible factors from the lamina may be phytohormones, and that the establishment of an endogenous phytohormone gradient in the explants may affect shoot bud differentiation in this culture system. Received: 7 April 1998 / Revision received: 8 April 1998 / Accepted: 17 April 1998     

An efficient method for adventitious shoot regeneration from stem-segment explants of gypsophila

An efficient adventitious shoot regeneration procedure was developed for Gypsophila paniculata L. Using cultivar Arbel, shoot regeneration from the three upper internodes of the stem was monitored on MS media supplemented with different cytokinins (thidiazuron, benzyladenine, kinetin or zeatin) and an auxin (naphthaleneacetic acid). Thidiazuron was found to be the most efficient cytokinin, with up to 100% of the explants forming shoots, at an average of up to 19 shoots per explant being regenerated. The highest percentage of shoot formation was observed in the stem explants originating from the first internode, with all cytokinins tested. The adventitious origin of shoots regenerated from stem explants was confirmed by scanning electron microscopy. The regeneration procedure was found to be applicable to five other gypsophila cultivars (Perfecta, Golan, Gilboa, Flamingo and Tavor). Regenerating plants were successfully transferred to soil, and did not differ in flower color, size or shape from standard vegetatively propagated plants derived from cuttings. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

Rapid adventitious shoot regeneration from leaf explants of European birch

The goal of this research was to develop a rapid and efficient system for regenerating shoots from leaf explants of European birch, Betula pendula Roth. Single-node stem explants were established in culture, and microshoots were subcultured every 4 weeks through 12 subcultures. Leaves from glasshouse plants or subcultured shoots were excised from stems, cut into approximately 35-mm² pieces, and placed on Woody Plant Medium (WPM) containing different combinations of naphthaleneacetic acid (NAA) (0, 3, 6 or 9 M) and benzyladenine (BA) (0, 7.5, 15 or 22.5 M) in a 4×4 factorial design. The percentage of leaf pieces forming shoots and the number of shoots regenerated per explant were recorded after 4 weeks. Only media containing BA without NAA stimulated shoot formation on leaf explants. Fifteen micromolar BA induced the most shoots to form on leaf explants compared to 30, 45 or 60 M of this cytokinin. In addition, shoot regeneration was enhanced up to four-fold between the first and eleventh subculture. Over 90% of the leaf explants regenerated shoots with an average of 18 buds formed per explant for the eleventh subculture. Almost twice as many explants formed shoots if their adaxial side was in contact with the medium rather than oriented away from it. The ability to regenerate shoots from leaves varied among plants, regardless of stock plant age. This reliable shoot regeneration system can be used for rapid shoot proliferation and potentially for genetic engineering of European birch.     

Comparative adventitious shoot induction in kentucky coffeetree root and petiole explants treated with thidiazuron and benzylaminopurine

Summary Adventitious shoot induction and elongation was compared between root and petiole explants of Kentucky coffeetree (Gymnocladus dioicus L.) explants treated with a factorial combination of benzylaminopurine (BA) and thidiazuron (TDZ). Petiole explants initiated more adventitious shoots compared to root explants. Up to 83% of petiole explants initiated shoots compared to 67% of root explants. Maximal shoot induction was approximately 12 or five shoots per responding explant for petiole and root explants, respectively. For both explant types, TDZ was more effective than BA for shoot induction. There was an interaction between BA and TDZ on shoot induction in petiole explants, with the greatest percentage of explants forming shoots and the highest number of shoots initiated on the combination of 0.5 μM TDZ plus 10μM BA and 1.0μM TDZ plus 5 or 10 μM BA. In contrast, increasing concentrations of BA inhibited shoot initiation in root explants with and without TDZ. While BA inhibited shoot initiation in root explants, it promoted shoot initiation in petiole explants. In contrast, TDZ was equally effective at inducing shoots in root and petiole explants. This suggests that root and petiole explants of Kentucky coffeetree could be a useful model system for studying the differences, in apparent mode of action between TDZ and BA on adventitious shoot initiation.     

Direct somatic embryogenesis and plant regeneration from leaf, petiole, and stem explants of Golden Pothos

Somatic embryos directly formed at cut edges or on the surface of leaf explants, around cut ends or along side surfaces of petiole and stem explants of Golden Pothos [Epipremnum aureum (Linden & Andre) Bunt.] on Murashige and Skoog (MS) medium supplemented with N-(2-chloro-4-pyridyl)-N-phenylurea (CPPU) or N-phenyl-N-1, 2, 3-thiadiazol-5-ylurea (TDZ) with -naphthalene acetic acid (NAA) and a medium called MK containing MS salts with Kaos vitamins, supplemented with 2.0 mg/l TDZ and 0.2 mg/l NAA. Somatic embryos were also produced on MS medium containing 2.0 mg/l kinetin (KN) and 0.5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) from leaf and petiole explants, MS medium supplemented with 2.0 mg/l CPPU and 0.5 mg/l 2,4-D from petiole and stem explants, and 2.0 mg/l TDZ and 0.2 mg/l or 0.5 mg/l 2,4-D from stem explants. In addition, somatic embryos occurred from stem explants on Chus N₆ medium containing 2.0 mg/l CPPU and 0.2 mg/l NAA. Somatic embryos matured and grew into multiple buds, shoots, or even plantlets after 2–3 months on the initial culture medium. Germination was optimal on MS medium containing either 2 mg/l 6-benzylaminopurine (BA) and 0.2 mg/l NAA or 2 mg/l zeatin and 0.2 mg/l NAA. Shoots elongated better and roots developed well on MS medium with no growth regulators. Approximately 30–100 plantlets were regenerated from each explant. The regenerated plants grew vigorously after transplanting to a soil-less container substrate in a shaded greenhouse.     

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.     

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.     

Direct regeneration of Drosera from leaf explants and shoot tips

An efficient protocol for the micropropagation of Drosera anglica, D. binata and D. cuneifolia is described. Proliferation was obtained from leaf segments and shoot tips, which served as initial explants. The regeneration capacity of explants was influenced by factors such as nutrient media, concentrations of growth regulators and the type of medium (liquid or solid). The highest number of plants regenerating from D. binata explants was obtained on the growth regulator-free Vacin and Went medium. In the case of D. anglica the highest proliferation rate was obtained on the Fast medium supplemented with 0.05 M 6-benzyladenine (BA) and 0.005 M -naphthaleneacetic acid (NAA), whereas for D. cuneifolia the optimal regeneration medium proved to be 1/2 MS with the growth regulator supplementation estimated at 0.2 M BA and 0.2 M NAA. Liquid media significantly increased the regeneration potential of D. anglica and D. binata explants.     

Effect of thidiazuron on adventitious shoot regeneration from seedling explants of Nothapodytes foetida

Summary Regeneration of adventitious shoots from the medicinal plant Nothapodytes foetida (Weight) Sleumer Syn. Mappia foetida (family Ieacinaceceae) has been achieved using different seedling explants. Direct, regeneration of shoot buds was observed in Murashige and Skoog's (MS) basal medium supplemented with various concentrations of thidiazuron. The optimum levels of thidiazuron concentrations were 0.91–4.45 μM. Leaf explants formed more shoots followed by hypocotyls or cotyledons. The shoot buds elongated and rooted on MS basal medium with N⁶-benzyladenine (0.88–2.22 μM) and indole-3-butyric acid (0.49 μM).     

Plant regeneration through the direct induction of shoot buds from petiole explants of Jatropha curcas: a biofuel plant

An efficient and reproducible method for the regeneration of Jatropha curcas plants has been developed. The method employed direct induction of shoot buds from petiole explants, without the formation of an intervening callus using a Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ). The best induction of shoot buds (58.35%) and the number of shoot buds per explant (10.10) were observed when in vitro petiole explants were placed horizontally on MS medium supplemented with 2.27 µM TDZ after 6 weeks. The induced shoot buds were transferred to MS medium containing 10 µM kinetin (Kn), 4.5 µM 6-benzyl aminopurine (BAP) and 5.5 µM α-naphthaleneacetic acid (NAA) for shoot proliferation. The proliferated shoots could be elongated on MS medium supplemented with different concentrations and combinations of BAP, indole-3-acetic acid (IAA), NAA and indole-3-butyric acid (IBA). MS medium supplemented with 2.25 µM BAP and 8.5 µM IAA was found to be the best combination for shoot elongation and 3.01–3.91 cm elongation was achieved after 6 weeks. However, significant differences in plant regeneration and shoot elongation were observed among the genotypes studied. The orientation (horizontal or vertical) and source (in vitro or in vivo) of explants also significantly influenced plant regeneration. The elongated shoots could be rooted on half-strength MS medium supplemented with 2% sucrose, different concentrations and combinations of IBA, IAA and NAA, and 0.25 mg L⁻¹ activated charcoal. Half-strength MS medium supplemented with 2% sucrose, 15 µM IBA, 5.7 µM IAA, 5.5 µM NAA and 0.25 mg L⁻¹ activated charcoal was found to be the best for promoting rooting. The rooted plants could be established in soil with more than 90% survival.     

In vitro shoot regeneration from cotyledons of immature ovules ofImpatiens platypetala Lindl.

Summary An efficient and reproducible protocol has been developed for in vitro shoot regeneration from cotyledonary explants derived by germinating immature ovules ofImpatiens platypetala Lindl. &#x2018;TR6-27-2&#x2019;. Cotyledonary explants were cultured on a modified Murashige and Skoog (MS) agar-solidified medium containing 7.5g &#x00B7; liter^&#x2212;1 sucrose, 22.2&#x00B5; M N⁶-benzyladenine (BA), and 0.54&#x00B5;M &#x03B1;-naphthaleneacetic acid (NAA). The induction of organogenic tissues occurred after 6 to 8 wk in culture. Exogenous auxin and cytokinin were essential for the induction of organogenic tissues and survival of explants, and BA was most effective for the induction of organogenic tissues, compared with other cytokinins tested. The addition of glutamine (500 mg &#x00B7; liter^&#x2212;1) was also important for growth of organogenic tissues after induction and for reducing explant death during culture. The induction of organogenic tissue was also influenced by the type of cotyledon cultured and the age of the donor seedlings. On average, eight shoots per explant were induced from organogenic tissues larger than 0.5 cm in diameter 6 to 8 wk after transfer to a modified MS agar-solidified medium without NAA and BA reduced to 4.44&#x00B5;M. Shoots longer than 0.5 cm in length were successfully rooted 2 to 4 wk after transfer to a basal MS medium containing 30g &#x00B7; liter^&#x2212;1 sucrose.     

Long-term multiplication of onion (Allium cepa L.) by cyclic shoot regeneration in vitro

We succeeded in cultivating onion plants in vitro with a high potential for shoot regeneration. The apex must be destroyed or injured to obtain axillary buds. This capacity was restricted to the abaxial base of the youngest sheaths. It was shown necessary to restore plant individuality before further proliferation; this process constituted one cycle. For successive regeneration each cycle was composed of three steps: shoot proliferation in the presence of a cytokinin, shoot individualization and plant development in the absence of growth regulators. Effect of growth regulators on the physiological status of onion plants cultured in vitro is discussed.Abbreviations BA 6-benzyladenine - NAA naphthaleneacetic acid