<Emphasis Type="Italic">In vitro</Emphasis> regeneration of the tennessee coneflower (<Emphasis Type="Italic">Echinacea tennesseensis</Emphasis>)

Summary Tennessee coneflower [Echinacea tennesseensis (Beadle) Small] was regenerated from flower stalks, leaf sections from flowering plants, and hypocotyls and cotyledons from seedlings. Murashige and Skoog medium (MS) supplemented with naphthaleneacetic acid (NAA) at 0.54 μM and thidiazuron (TDZ) at 22.7 μM yielded the most shoots per leaf explant. NAA and 6-benzylaminopurine concentrations for optimal shoot regeneration from leaf, flower stalk, cotyledon and hypocotyl explants in MS media were 0.54 and 24.6μM, respectively. All explant types generated shoots; however, those derived from leaves and flower stalks produced the highest number of shoots per explant and highest percentage of explants with shoots. Explants cultured on media containing high levels of NAA (5.4–27 μM) formed calluses but no adventitious shoot. Leaf explants responded to a wider range of NAA concentrations than the other explant types but shoots generated from flower stalks grew the fastest. While all cytokinins tested increased the number of shoots per explant, the number of shoots in media containing TDZ was increased by nearly threefold. Regenerated shoots from all explant types cultured on MS medium supplemented with 0.25 μM indole-3-butyric acid initiated roots within 4 wk; NAA was not effective for root induction. All vernalized plantlets developed into plants that were morphologically identical to the source material.     

Efficient procedures for callus induction and adventitious shoot organogenesis in sugar beet (<Emphasis Type="Italic">Beta vulgaris</Emphasis> L.) breeding lines

Summary Improved in vitro tissue culture systems are needed to facilitate the application of transgene technology to the improvement of sugar beet germplasms. Several commercially important sugar beet breeding lines (SDM, 3, 5, 8, 9, 10, 11, HB 526, and CMS 22003) and commercial varieties (Roberta and Gala) were tested for their regeneration capacity through adventitious shoot organogenesis from cotyledons, hypocotyls, root/hypocotyl/shoot transition zone tissues, and leaf lamina and petiole via an intervening callus phase. Callus induction and adventitious shoot regeneration was dependent on genotype and combinations of plant growth regulators. With cotyledon or hypocotyl explants, SDM 3 and 10 showed a better response on adventitious shoot regeneration in medium containing benzyladenine (BA) and 2,3,5-triiodobenzoic acid or 1-naphthaleneacetic acid (NAA) than SDM 11, 5, and 9. Shoot regeneration was obtained from hypocytyl-root or hypocotyl-shoot transition zone tissue in SDM 9, 10, and HB 526 grown on PGo medium supplemented with BA to induce callus, and the regeneration frequency was 25%. Adventitious shoots were also regenerated from leaf explants of SDM 3 and 9 cultured on medium containing NAA for callus induction and BA and NAA to induce shoot regeneration, and in SDM 10 and CSM 22003 cultured on medium containing BA for callus induction and to induce shoot regeneration.     

High frequency organogenesis in hypocotyl,cotyledon, leaf and petiole explants of broccoli (Brassica oleracea L. var. italica), an important vegetable crop

Broccoli (Brassica oleracea L. var. italica) is an important, nutritionally rich vegetable crop, but severely affected by environmental stresses, pests and diseases which cause massive yield and quality losses. Genetic manipulation is becoming an important method for broccoli improvement. In the present study, a reproducible and highly efficient protocol for obtaining organogenesis from hypocotyl, cotyledon, leaf and petiole explants of broccoli (Brassica oleracea L. var. italica cv. Solan green head) has been developed. Hypocotyl and cotyledon explants were used from 10 to 12 days old aseptically grown seedlings whereas leaf and petiole explants were excised from 18 to 20 days old green house grown seedlings and surface sterilized. These explants were cultured on shoot induction medium containing different concentration and combination of BAP and NAA. High efficiency shoot regeneration has been achieved in hypocotyl (83.33 %), cotyledon (90.11 %), leaf (62.96 %) and petiole (91.10 %) explants on MS medium supplemented with 3.5 mg/l BAP + 0.019 mg/l NAA 2.5 mg/l BAP + 0.5 mg/l NAA, 4.0 mg/l BAP + 0.5 mg/l NAA and 4.5 mg/l BAP + 0.019 mg/l NAA respectively. Petiole explants showed maximum shoot regeneration response as compared to other explants. MS medium supplemented with 0.10 mg/l NAA was found best for root regeneration (100 %) from in vitro developed shoots. The regenerated complete plantlets were transferred to the pots containing cocopeat and successfully acclimatized. This optimized regeneration protocol can be efficiently used for genetic transformation in broccoli. This is the first comparative report on multiple shoot induction using four different types of explants viz. hypocotyl, cotyledon, leaf and petiole.     

<Emphasis Type="Italic">In vitro</Emphasis> organogenesis and antioxidant enzymes activity in <Emphasis Type="Italic">Acanthophyllum sordidum</Emphasis>

The effect of various hormonal combinations on callus formation and regeneration of shoot and root from leaf derived callus of Acanthophyllum sordidum Bunge ex Boiss. has been studied. Proteins and activity of antioxidant enzymes were also evaluated during shoot and root organogenesis from callus. Calli were induced from leaf explants excised from 30-d-old seedlings grown on Murashige and Skoog medium containing 4.52 μM 2,4-dichlorophenoxyacetic acid + 4.65 μM kinetin. Maximum growth of calli and the most efficient regeneration of shoots and roots occurred with 2.69 μM 1-naphthalene acetic acid (NAA), 2.69 μM NAA + 4.54 μM thidiazuron and 2.46 μM indole-3-butyric acid. Protein content decreased in calli and increased significantly during regeneration of shoots from callus. Superoxide dismutase activity decreased in calli comparing to that of seedlings, then increased in regenerated shoots and roots. High catalase activity was detected in seedlings and regenerated shoots, whereas high peroxidase activity was observed in calli and regenerated roots.     

Direct shoot organogenesis and assessment of genetic stability in regenerants of <Emphasis Type="Italic">Solanum aculeatissimum</Emphasis> Jacq.

A simple and efficient procedure was developed for in vitro propagation of Solanum aculeatissimum Jacq. using leaf and petiole explants cultured on Murashige and Skoog (MS) medium supplemented with α-naphthalene acetic acid (NAA) and 6-benzyladenine (BA). Effects of various plant growth regulators, explant types, carbohydrates, and basal salts on induction of adventitious shoots were also studied. Leaf explants appeared to have better regeneration capacity than petiole explants in the tested media. The highest regeneration frequency (79.33 ± 3.60%) and shoot number (11.33 ± 2.21 shoots per explant) were obtained in leaf explants in MS medium containing 3% sucrose and 0.8% agar, supplemented with 0.1 mg/l NAA and 2.0 mg/l BA, whereas petiole explants were more responsive to 0.1 mg/l NAA and 1.0 mg/l thiadiazuron. Developed shoots rooted best on MS medium with 1.0 mg/l indole acetic acid (IAA), producing 18.33 ± 2.51 roots per shoot. Histological investigation showed that the shoot buds originated mainly from epidermal cells of wounded tissues, without callus formation. The regenerated plantlets were successfully acclimatized in a greenhouse, where over 90% developed into morphologically normal and fertile plants. Results of flow cytometry analysis on S. aculeatissimum indicated no variation in the ploidy levels of plants regenerated via direct shoot formation and showed almost the same phenotype as that of mother plants. This adventitious shoot regeneration method may be used for large-scale shoot propagation and genetic engineering studies of S. aculeatissimum.     

A two-stage pretreatment of seedlings improves adventitious shoot regeneration in sugar beet (<Emphasis Type="Italic">Beta vulgaris</Emphasis> L.)

The effects of a two-stage pretreatment of seedlings on the subsequent shoot regeneration capacity were investigated. Pretreated seedlings were obtained by germinating seeds on three different germination media and then further culturing on six different growth media. Lamina and petiole explants of two sugar beet (Beta vulgaris L.) breeding lines were then excised from the pretreated seedlings and cultured on five different shoot regeneration media. In both breeding lines, petiole explants produced significantly more shoots than lamina explants with higher frequencies of organogenic capacities; petiole explants of the lines M1195 and ELK345 produced a mean of 2.1 and 2.7 shoots per explant while their lamina explants produced 1.5 and 2.2 shoots per explant, respectively. A genotypic variation was evident as the line ELK345 was more productive for shoot development from both types of explants. In overall comparisons of different germination, growth and regeneration media, germination medium was most effective when supplemented with 0.5 mg/l 6-benzyladenine (BA) while both growth and regeneration media were most productive when contained a combination of 0.25 mg/l BA and 0.10 mg/l indole-3-butyric acid (IBA). Of all the treatments tested, the highest mean number of shoots per explant (8.3 shoots) and frequency of organogenic explants (75.6%) were obtained on regeneration medium supplemented with 0.25 mg/l BA and 0.10 mg/l IBA when petiole explants of the line ELK345 were excised from the seedlings that had been germinated on medium containing 0.5 mg/l BA followed by further growth on medium containing 0.25 mg/l BA and 0.10 mg/l IBA.     

Thidiazuron-induced high-frequency axillary and adventitious shoot regeneration in Vigna radiata (L.) Wilczek

Summary Prolific shoot regeneration was achieved in mungbean Vigna radiata (L.) Wilczek from 3-d-old in vitro cotyledonary node and hypocotyl explants from seedlings derived from mature seeds on Murashige and Skoog (MS) medium supplemented with thidiazuron (TDZ) (0.9 μM). An initial exposure to TDZ for 20 d and three successive transfers to fresh medium with reduced thidiazuron levels (0.09 μM) resulted in the regeneration of 104 shoots/explant from the cotyledon and 30 shoots/explant from the hypocotyl. Thidiazuron-associated abnormalities such as short compact shoots, fasciation and leaf growth in the form of rosettes were observed in shoots regenerated from hypocotyl explants. Both axillary and adventitious shoot formation from the explants were confirmed by histology. Through repectitive cycles of regeneration in the presence of TDZ, the number of shoots that could be obtained from the two explant classes within 80 d was significantly higher than with previous reports in mungbean     

Efficient plant regeneration from cotyledon explants of bottle gourd (<Emphasis Type="Italic">Lagenaria siceraria</Emphasis> Standl.)

Using cotyledon explants excised from seedlings germinated in vitro, an efficient plant regeneration system via organogenesis was established for bottle gourd (Lagenaria siceraria Standl.). Maximum shoot regeneration was obtained when the proximal parts of cotyledons from 4-day-old seedlings were cultured on MS medium with 3 mg/l BA and 0.5 mg/l AgNO₃ under a 16-h photoperiod. After 3–4 weeks of culture, 21.9–80.7% of explants from the five cultivars regenerated shoots. Adventitious shoots were successfully rooted on a half-strength MS medium with 0.1 mg/l IAA for 2–3 weeks. Flow cytometric analysis revealed that most of the regenerated plants derived from culture on medium with AgNO₃ were diploid.     

A shoot regeneration protocol effective on diverse genotypes of sunflower (Helianthus annuus L.)

Summary We describe a protocol, and several experiments that helped lead to its development, for sunflower regeneration. Important factors for sunflower regeneration were explant age, cytokinin type and concentration, basal medium, and explant source. We could not induce shoot regeneration from the explants derived from mature tissues including leaf, petiole, and stem. However, use of juvenile explants such as embryo meristem and primordial leaf tissues allowed routine regeneration of 17 different sunflower genotypes. High frequency of shoot regeneration was achieved with these explants taken from seedlings up to 5 d after germination. Explant age was less critical for embryo meristem explants than for primordial leaf tissues. Of the four basal media tested, MS and B5 media produced higher shoot-regeneration frequencies than did Anderson and woody plant media. The highest shoot-regeneration frequency was obtained with MS medium supplemented with 2 μM BA and without auxin. Addition of 1 μM naphthalene-acetic acid to the medium significantly reduced both the percentage of explants producing shoots and average number of shoots per explant. Regenerated shoots were grown to maturity in a greenhouse.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of <Emphasis Type="Italic">Echinacea pallida</Emphasis> from leaf explants

Summary A method has been developed for the induction of adventitious shoots from leaf tissue of Echinacea pallida with subsequent whole-plant regeneration. Proliferating callus and shoot cultures were derived from leaf tissue explants placed on Murashige and Skoog medium supplemented with 6-benzylaminopurine and naphthaleneacetic acid combinations. The optimum shoot regeneration frequency (63%) and number of shoots per explant (2.3 shoots per explant) was achieved using media supplemented with 26.6 μM 6-benzylaminopurine and 0.11 μM naphthaleneacetic acid. Rooting of regenerated shoot explants was successful on Murashige and Skoog medium, both with and without the addition of indole-3-butyric acid. All plantlets survived acclimatization, producing phenotypically normal plants in the greenhouse. This study demonstrates that leaf tissue of E. pallida is competent for adventitious shoot regeneration and establishes a useful method for the micropropagation of this important medicinal plant.     

Identification of highly regenerative plants within sugar beet (<Emphasis Type="Italic">Beta vulgaris</Emphasis> L.) breeding lines for molecular breeding

Summary Development of an efficient transformation method for recalcitrant crops such as sugar beet (Beta vulgaris L.) depends on identification of germplasm with relatively high regeneration potential. Individual plants of seven sugar beet breeding lines were screened for their ability to form adventitious shoots on leaf disk callus. Disks were excised from the first pair of true leaves of 3-wk-old seedlings or from partially expanded leaves of 8-mo.-old plants and cultured on medium with 4.4 μM 6-benzylaminopurine for 10 wk. At 5 wk of culture, friable calluses and adventitious shoots began to develop. Rates of callus and shoot formation varied between breeding lines and between individual plants of the same line. Line FC607 exhibited the highest percentage (61%) of plants that regenerated shoots on explants. Among the plants with a positive shoot regeneration response, line FC607 also had the highest mean number (8.3±1.1) of shoots per explant. Individual plants within each line exhibited a wide range of percentages of explants that regenerated shoots. A similar variation was observed in the number of shoots that regenerated per explant of an individual plant. No loss of regeneration potential was observed on selected plants maintained in the greenhouse for 3 yr. Regenerated plants exhibited normal phenotypes and regeneration abilities comparable to the respective source plants. Based on our results, it is imperative to screen a large number of individual plants within sugar beet breeding lines in order to identify the high regenerators for use in molecular breeding and improvement programs.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of an endangered plant species, <Emphasis Type="Italic">Thermopsis turcica</Emphasis> (Fabaceae)

This report deals with micropropagation of the critically endangered and endemic Turkish shrub, Thermopsis turcica using callus, root and cotyledonary explants. Callus cultures were initiated from root and cotyledon explants on MS medium supplemented with 0.5–20 μM NAA or 2,4-D. The root explants were found to be better in terms of quick responding and callusing percentages as compared to the cotyledons. Organogenic callus production with adventitious roots and shoots were obtained on MS medium with only NAA. The calli obtained with NAA, root and cotyledonary explants were cultured with BA and kinetin (2–8 μM) alone or in combination with a low level (0.5 μM) of 2,4-D or NAA. The best regeneration of shoots from root explants was observed on hormone-free MS medium. NAA with BA or kinetin in the medium improved shoot induction from the calli obtained with NAA. Maximum percentage of shoots (93.3%), maximum number of shoots (6.2) and maximun length of shoots (8.22 cm) were achieved from cotyledonary explants at 4 μM BA and 0.5 μM NAA. The presence of 0.5 μM or higher levels of 2,4-D in shoot induction medium inhibited the regeneration in T. turcica explants. 83% of in vitro rooting was attained on pulsed-IBA treated shoots. The regenerated plants with well developed shoots and roots were successfully acclimatized. Application of this study’s results has the potential to conserve T. turcica from extinction.     

<Emphasis Type="Italic">Organogenic callus</Emphasis> as the target for plant regeneration and transformation via <Emphasis Type="Italic">Agrobacterium</Emphasis> in soybean (<Emphasis Type="Italic">Glycine max</Emphasis> (L.) Merr.)

A regeneration and transformation system has been developed using organogenic calluses derived from soybean axillary nodes as the starting explants. Leaf-node or cotyledonary-node explants were prepared from 7 to 8-d-old seedlings. Callus was induced on medium containing either Murashige and Skoog (MS) salts or modified Finer and Nagasawa (FNL) salts and B5 vitamins with various concentrations of benzylamino purine (BA) and thidiazuron (TDZ). The combination of BA and TDZ had a synergistic effect on callus induction. Shoot differentiation from the callus occurred once the callus was transferred to medium containing a low concentration of BA. Subsequently, shoots were elongated on medium containing indole-3-acetic acid (IAA), zeatin riboside, and gibberellic acid (GA). Plant regeneration from callus occurred 90 ∼ 120 d after the callus was cultured on shoot induction medium. Both the primary callus and the proliferated callus were used as explants for Agrobacterium-mediated transformation. The calluses were inoculated with A. tumefaciens harboring a binary vector with the bar gene as the selectable marker gene and the gusINT gene for GUS expression. Usually 60–100% of the callus showed transient GUS expression 5 d after inoculation. Infected calluses were then selected on media amended with various concentrations of glufosinate. Transgenic soybean plants have been regenerated and established in the greenhouse. GUS expression was exhibited in various tissues and plant organs, including leaf, stem, and roots. Southern and T₁ plant segregation analysis of transgenic events showed that transgenes were integrated into the soybean genome with a copy number ranging from 1–5 copies.     

A rapid and efficient in vitro shoot regeneration protocol using cotyledons of London plane tree (<Emphasis Type="Italic">Platanus acerifolia</Emphasis> Willd.)

A rapid, prolific and reproducible protocol for in vitro shoot regeneration from mature cotyledons of Platanus acerifolia has been developed. The influences of different plant growth regulator (PGR) combinations and donor seedling ages on shoot regeneration were investigated. The results showed that the application of BA in conjunction with NAA was the most effective PGR combination for the induction of shoot regeneration. When cotyledon explants of 5-day-old seedlings were incubated on MS basal medium supplemented with 4.0 mg L^?1 BA and 0.2 mg L^?1 NAA, 67.6?±?4.9% of the cotyledon segments produced adventitious shoots. These regenerated shoots were initially formed as stunted rosette cluster forms and were encouraged to elongate to produce distinct shoots by transfer onto MS medium containing 0.5 mg L^?1 BA and 0.05 mg L^?1 NAA; the resulting mean number of adventitious shoots per explant was 5.81?±?0.36. The elongated shoots were readily induced to root (i.e. 89.3% of shoots) by incubation on ½-strength MS medium supplemented with 0.1 mg L^?1 IBA. This is the first report of an efficient in vitro shoot regeneration protocol for P. acerifolia through direct organogenesis using cotyledon explants. Hence, this provides a more efficient basis for the Agrobacterium-mediated genetic transformation of Platanus than previously available.     

Callus formation and plant regeneration in various <Emphasis Type="Italic">Lilium</Emphasis> species and cultivars

Summary In researching the application of genetic transformation to lily breeding, callus formation from cultured explants and plant regeneration from induced calluses were examined in 33 Lilium genotypes, 21 species, three Asiatic hybrids, two LA hybrids, two Longiflorum hybrids, three Oriental hybrids, and two Trumpet hybrids. Seed, bulb scale, leaf, or filament explants were placed on a medium containing 4.1 μM 4-amino-3,5,6-trichloropicolinic acid (picloram; PIC) and cultured in the dark. After 2 mo., callus formation was observed in 30 genotypes, and a formation frequency of more than 50% was obtained in 24 genotypes. Bulb scale and filament explants showed great ability to form calluses, whereas seeds had poor ability. Most of the induced calluses were yellow and had a nodular appearance. When subcultured onto the same fresh medium, twofold or more increases in callus mass were obtained in 1 mo. for 15 genotypes. Callus lines showing sustained growth 1 yr after the initiation of subculture were examined for their ability to produce shoots on a medium without plant growth regulators (PGRs) and a medium containing 22 μM 6-benzyladenine (BA). Shoot regeneration was observed in all genotypes examined, and a regeneration frequency of over 80% was obtained in 20 genotypes. Initial explants used for callus induction and callus type (nodular or friable) had no effect on shoot regeneration. Most of the regenerated shoots developed into complete plantlets following their transfer to a PGR-free medium.     

TDZ-induced high frequency plant regeneration through multiple shoot formation in witloof chicory (<Emphasis Type="Italic">Cichorium intybus</Emphasis> L.)

A very efficient and rapid regeneration system via multiple shoot formation was developed for Cichorium intybus L. when leaf explants excised from sterile seedlings were cultured on medium supplemented with different concentrations and combinations of various plant growth regulators. In a comparison of leaf lamina and petiole explants, lamina explants produced over three times more shoots than petiole explants, with a mean of 7.5 shoots compared to 2.4. Of the combinations of KIN/IAA, KIN/NAA, BAP/IAA, or BAP/NAA, 0.5 mg l⁻¹ KIN combined with 0.3 mg l⁻¹ IAA was the most effective, producing a mean of 19.7 shoots per lamina explant while the control treatment involving no plant growth regulators produced no shoots at all. When either cytokinin was used alone, BAP was found nearly twice more successful than KIN. However, the most effective treatment of all was the combination of 0.01 mg l⁻¹ TDZ and 1.0 mg l⁻¹ IAA, producing as many as 35.8 shoots per lamina explant. This rate of shoot regeneration is remarkably higher than those previously reported for C. intybus, most likely due to the highly inductive effect of TDZ, which was tested for the first time in this species. Rooting of the shoots was readily achieved on medium containing different concentrations of IAA or IBA. IAA was more effective than IBA and resulted in the highest frequency of shoots that rooted (100%) and mean number of roots per shoot (4.2) when used at 0.5 mg l⁻¹. Hardening off process resulted in a production of more than 80% healthy plantlets.     

High-frequency regeneration and transformation ofRaphanus savus

We have achieved high-frequency shoot regeneration in radish(Raphanus sativus). Cotyledon explants from four-day-old seedlings were suitable for the effective induction of shoots on Murashige and Skoog’s (MS) medium containing 3.0 mg/L kinetin. We also determined that it was essential to include 1- to 2-ram petiole segments with the cotyledons for efficient induction. When the regenerated shoots were transferred to an MS liquid medium containing 0.1 mg/L NAA, roots formed within four weeks, and normal plant development ensued. We established a transformation protocol using anAgrobacterium binary vector that carries the GUS reporter gene. Preculturing the explants for I d in an MS medium containing 3.0 mg/L kinetin also increased efficiency. Five days of cocultivation proved best for delivering T-DNA into radish. Transformation frequencies of up to 52% were obtained in shoot induction media that contained 3.0 mg/L kinetin.     

<Emphasis Type="Italic">In vitro</Emphasis> adventitious shoot organogenesis and plant regeneration from seedling explants of <Emphasis Type="Italic">Albizia odoratissima</Emphasis> L.f. (Benth.)

Epicotyl, petiole, and cotyledon explants derived from 14-d-old seedlings of Albizia odoratissima were cultured on Murashige and Skoog (MS) basal medium supplemented with different concentrations of either 6-benzylaminopurine (BAP) solely or in combination with 0.5 μM naphthalene-3-acetic acid (NAA). The percentage of shoot regeneration and the number of shoots regenerated varied significantly depending on the type of explants used, the concentration of plant growth regulators, and the orientation of explants on the culture medium. The best response in terms of the percentage of shoot regeneration was obtained from epicotyls cultured horizontally on MS medium supplemented with 5 μM BAP, whereas the highest number of shoots per responding explant was recorded on medium containing 2.5 μM BAP and 0.5 μM NAA. Successful rooting was achieved by placing the microshoots onto MS medium containing 25 μM indole-3-butyric acid (IBA) for 24 h first, then transferring to the same medium without IBA. Of the various substrates tested, vermiculite was the best for plant acclimatization, as 75% of the plants survived and became established.     

In vitro plant regeneration from seedling-derived explants of ramie [Boehmeria nivea (L.) Gaud]

Ramie [Boehmeria nivea (L.) Gaud] is one of the most important perennial fiber crops in China. In vitro tissue culture of ramie could serve as an important means for its improvement through genetic transformation. To improve the regeneration capacity of ramie, the effects on plant regeneration of donor plant age, basal medium, plant growth regulators, and culture conditions were evaluated using explants derived from the cotyledon, hypocotyl, leaf, petiole, and stem of ramie seedlings. Cotyledons and hypocotyls excised from 4-d-old seedlings and leaves and petioles and stems from 15-d-old seedlings were optimal explants. The highest regeneration efficiency was obtained on Murashige and Skoog salts with Gamborg’s B₅ vitamins basal medium containing 2.27 μM thidiazuron (TDZ) and 0.054 μM naphthaleneacetic acid (NAA) for the five explant types tested. A photoperiod of 16:8 h (light/dark) was found to be superior than continuous darkness for regeneration of ramie using TDZ. The regenerated shoots were transferred to hormone-free medium for shoot elongation and successfully rooted on half-strength Murashige and Skoog supplemented with 0.134 μM NAA. The rooted plantlets with four to five leaves were transplanted to greenhouse for further growth.     

Pluronic F‐68 Enhanced Shoot Regeneration in Micropropagated Citrus Rootstock and Passiflora Species

The promotory effects have been studied of the non‐ionic surfactant, Pluronic F‐68, on bud induction/shoot regeneration in epicotyl and cotyledon explants of Citrus depressa and on shoot regeneration from leaf segments of 4–6 week‐old axenic nodal segment‐derived in vitro plants of Passiflora mollissima, P. giberti and P. edulis var. flavicarpa. For epicotyls of C. depressa, supplementation of agar‐solidified MS‐based bud induction/shoot regeneration medium with 0.5% [w/v] Pluronic F‐68 significantly (P < 0.05) increased mean fresh weight gain of cultures, percentage of explants giving shoots and number of shoots per explant. The same Pluronic concentration also enhanced the mean percentage of cotyledons exhibiting bud induction and the number of buds regenerated per cotyledon explant. Fresh weight gain was unaffected across the range of concentrations (0.001–0.5% w/v) of Pluronic F‐68 evaluated for this latter explant source. For leaf explants from axenic shoot cultures of P. mollissima, supplementation of NN‐based medium, containing 3 mg/l 6‐benzyladenine and 2.0 mg/l kinetin with 0.001–0.5% [w/v] Pluronic F‐68, significantly (P < 0.05) increased mean (± s.e.m.) biomass gain by a maximum of 2.7 ± 0.1 g fresh weight (g f.wt.) over the control. Similarly, for leaf explants of P. giberti, 0.001–0.5% [w/v] Pluronic F‐68 in MS‐based medium, containing 1.0 mg/l 6‐BAP and 0.5 mg/l kinetin significantly (P < 0.05) increased mean percentage of explants undergoing shoot regeneration. For P. edulis leaf explants, mean f.wt. gain was also significantly (P < 0.05) higher with Pluronic F‐68 at 0.001–0.5% [w/v].