<Emphasis Type="Italic">In vitro</Emphasis> regeneration of <Emphasis Type="Italic">Salvia nemorosa</Emphasis> L. from shoot tips and leaf explants

Summary Shoot tips and leaves excised from in vitro shoot cultures of Salvia nemorosa were evaluated for their organogenic capacity under in vitro conditions. The best shoot proliferation from shoot tips was obtained on Murashige and Skoog (MS) medium supplemented with 8.9 μM 6-benzylaminopurine (BA) and 2.9 μM indole-3-acetic acid (IAA). Leaf lamina and petiole explants formed shoots through organogenesis via callus stage and/or directly from explant tissue. The highest values for shoot regeneration were obtained with 0.9 μM BA and 2.9 μM IAA for lamina explants. No shoot organogenesis was obtained on leaf explants cultured on MS medium supplemented with α-naphthaleneacetic acid (NAA). The regenerated shoots rooted the best on MS medium containing 0.6 μM IAA or 0.5 μM NAA. In vitro-propagated plants were transferred to soil with a survival rate of 85% after 3 mo.     

Thidiazuron promotes adventitious shoot regeneration from pothos (Epipremnum aureum) leaf and petiole explants

Summary Regeneration of adventitious shoots of pothos (Epipremnum aureum Linden and Andre) ‘Jade’ was obtained using leaf and petiole explants preprated from shoot tips of 3-yr-old greenhouse-grown plants. Explants were cultured on Murashige and Skoog (MS) basal medium supplemented with thidiazuron (TDZ), 6-(4-hydroxy-3-methy-trans-2-butenyl-amino)purine (zeatin) or N-isopentenylaminopurine (2iP) individually with α-naphthaleneacetic acid (NAA) in 18 combinations. Callus was initiated from cut surface and along the midrib or major vein of leaf sections. Shoot regeneration from leaf and petoole explants occurred in 30d on medium containing 1, 5 or 10μM TDZ with 0.5 or 1.0μM NAA except petioles on medium with 10 μM TDZ and 1.0 μM NAA where regeneration failed. More time (50d) was needed for shoot regeneration when explants were cultured on medium containing either 2iP or zeatin with NAA. Regeneration frequencies were up to 20% and 50% for leaf and petiole explants, respectively. Shoot numbers per responding explant attained 30 for leaf and petiole explants on medium containing TDZ but only one to four on medium containing either 2iP or zeatin. These results indicate that TDZ is a more effective cytokinin for in vitro regeneration of pothos than either zeatin or 2iP.. Shoots elongated readily and rooted well on MS basal medium, without plant growth regulators. Plantlets acclimatized rapidly and grew vigorously in the greenhouse after transfer to pots containing a commerecial potting medium.     

High-frequency adventitious shoot bud induction and shoot elongation of Chile pepper (Capsicúm annuum L.)

Summary In vitro plantlet regeneration was obtained from cultured cotyledon and young leaf explants of five Indian chile pepper cultivars (Capsicum annuum L. evs. Gujarat-1, Gujarat-2, Guntur-4, Selection-49, and Jwala). Adventitious shoot buds (ASB) were regenerated directly from cotyledon and young leaf explants in all the five cultivars on media containing benzyladenine (BA) alone or in combination with 1-naphthaleneacetic acid (NAA). Regeneration frequency was highly influenced by cultivar explant type, media combination and their interactions, except the interaction between cultivar and explant, for number of ASB per explant. Percent contribution of individual source suggested that selection of explant type followed by medium combination and cultivars was essential for obtaining high-frequency ASB induction. Across different cultivars the young leaf explant was found to be the most responsive explant, while Murashige and Skoog (MS) medium containing BA alone (17.8, 26.6, and 35.5 μM) was found to be the best medium for the production of maximum number of ASB. Between the two explants, shoot elongation was observed with ASB obtained from young leaf explants on MS medium containing BA (2.2 and 4.4 μM) and gibberellie acid (GA₃) (1.4, 2.9, 4.3 and 5.8 μM). The MS medium fortified with 4.4 μM BA+2.9μM GA₃ was optimum for shoot elongation. Elongated shoots were rooted on liquid MS medium supplemented with 2.9 μM indole-3-acetic acid (IAA) and successfully established ex vitro.     

Liquid culture for efficient micropropagation of Wasabia Japonica (MIQ.) matsumura

Summary An efficient protocol for in vitro propagation of the valuable medicinal plant, Wasabia japonica (Miq.) Matsumura is described through shoot tip proliferation and direct regeneration. Multiple shoots were induced from shoort tips cultured on Murashige and Skoog (MS) semi-solid medium containing various concentrations (0.5–50 μM) of N⁶-benzyladenine (BA), thidiazuron, kinetin, and zeatin. A comparison was made on shoot multiplication between semi-solid and liquid culture media. Well-developed shoots were obtained using full-strength MS semi-solid medium containing 5.0 μM BA. However, the greatest shoot proliferation was achieved on either full- or half-strength MS liquid media supplemented with 5.0 μM BA for 4 wk (15.3±0.9 and 15.0±0.7 shoots per explant, respectively), and on half-strength MS liquid medium for 6 wk (25.8±1.3 shoots per explant) in culture. In contrast, the maximum number of shoots per explant on full-strength MS semi-solid medium was achieved with either 5.0 μM BA (10.4±0.6 shoots per explant) or 10.0 μM kinetin (10.9±0.8 shoots per explant). Fresh weight of explants and length of shoots derived from full-strength MS liquid medium (1055±77 mg and 34.2±1.0 mm, respectively) were significantly higher than those derived from full-strength MS semisolid medium (437.6±17.3 mg and 15.4±0.7 mm, respectively). Quarter-strength MS liquid medium had no significant difference in shoot proliferation when compared to quarter-strength MS semi-solid medium. Elongated shoots were separated and rooted on half-strength MS semi-solid media fortified with 1-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), or indole-3-acetic acid (IAA) ranging from 0.1 to 10.0 μM. Root formation was greatest with IBA when compared with IAA and NAA. One hundred percent of shoots were rooted on half-strength MS medium with 5.0 μM IBA, while vigorous roots were obtained with 10.0 μM IBA. Micropropagated plantlets were successfully established in soil with 95% survival rate after heardening.     

Micropropagation of <Emphasis Type="Italic">Scabiosa caucasica</Emphasis> Bieb. Cv. Caucasica blue

Summary Micropropagation of Scabiosa caucasica cv. Caucasica Blue was achieved by culturing, separating axillary and adventitious shoots, or node sectioning on Murashige and Skoog (MS) medium supplemented with benzyladenine (BA). The highest frequency of adventitious shoots regenerated from nodal or internodal explants and leaf blade (with or without petiole) appeared to occur on MS medium with 4.4 and 18 μM BA, respectively. Addition of 0.19 or 1.9 μM α-naphthaleneacetic acid to the BA-containing medium promoted callus formation and reduced shoot organogenesis. During micropropagation, shoot nodal explants derived from in vitro shoots cultured on MS medium supplemented with 4.4 μM BA yielded 8.9 shoots per explant within 40 d after culture initiation.     

Direct regeneration from in vitro leaf and petiole tissues of <Emphasis Type="Italic">Populus tremula</Emphasis> ‘Erecta’

Dormant buds from a mature tree of Populus tremula ‘Erecta’ were incubated on a Murashige and Skoog (MS) medium supplemented with 1.0 μM thidiazuron (TDZ). Induced shoots were then proliferated on medium of MS or Woody Plant Medium (WPM), or Driver and Kuniyuki Walnut (DKW) supplemented with varying levels of benzyladenine (BA). Overall, shoots grown on MS medium supplemented with 1.25–2.5 μM BA exhibited the highest frequency of shoot proliferation (>95%) and more than 60% of responding explants produced more than five shoots per explant. Shoot organogenesis was induced from both leaf and petiole explants incubated on WPM medium containing BA, or TDZ, or zeatin. Among the different cytokinins tested, zeatin induced the highest frequency (average 72.1%) of shoot organogenesis. None of explants survived on media containing no cytokinins within 6–8 weeks following culture. Overall, a higher frequency of shoot regeneration was obtained from petioles than from leaf explants. The highest frequency of regeneration was achieved when petioles were incubated on WPM containing 10–20 μM zeatin. Addition of naphthaleneacetic acid (NAA) did not have a significant effect on shoot regeneration in all treatments. Shoot organogenesis was directly induced from petiole explants without intervening callus. Regenerated shoots were easily rooted on all tested media supplemented with 0.5 μM NAA. Rooted plants were transferred to potting mix and grown in the greenhouse.     

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

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

Direct shoot regeneration from leaf explants of <Emphasis Type="Italic">Rosa damascena</Emphasis> Mill.

Summary A protocol for in vitro propagation using direct induction of shoot buds from leaf explants of in vitro-raised shoots of Rosa damascena var. Jwala is reported. The present study is the first report on direct shoot regeneration in scented roses. Elite plants raised from nodal explants and maintained for over 2yr in vitro on a static liquid shoot multiplication Murashige and Skoog (MS) medium supplemented with 5.0 μM benzyladenine (BA) and 3% sucrose were used. Petioles from fully developed young leaves, obtained after 4 wk of pruning of old shoots, were found to be ideal for regeneration of shoots. Initially the explants were cultured in an induction medium [half-strength MS+3% sucrose+6.8μM thidiazuron+0.27 μM α-naphthaleneacetic acid (NAA)+17.7 μM AgNO₃] and subsequently transferred to the regeneration medium (MS+2.25 μM BA+0.054 μM NAA) after 7, 14, 21, 28, and 35d. The highest shoot regeneration response (69%) was recorded when shoots were kept in the induction medium for 21 d and later transferred to regeneration medium. Histological studies revealed direct formation of shoot buds without the intervening callus phase. In vitro rooting of micro-shoots was accomplished within 2wk on half-strength MS liquid medium supplemented with 10.0 μM IBA and 3% sucrose for 1 wk in the dark and later transferred to hormone-free medium and kept in the light. Plantlets, remaining in the latter medium for 5–6 wk when transferred to soil, showed 90% survival.     

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.     

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.     

Direct shoot regeneration from leaf and internode explants of Aloysia polystachya [GRIS.] mold. (Verbenaceae)

Summary Adventitious bud regeneration from leaf and internode explants of Aloysia polystachya was achieved. Shoots from nodal segments grown in vitro were cut into pieces and used as sources of explants. Organogenesis was induced from both explants cultured on quarter-strength Murashige and Skoog (MS) semisolid medium (plus sucrose 5 g l⁻¹) containing different combinations of 6-benzyladenine (BA) and α-naphthaleneacetic acid (NAA) under 116 μmol m⁻² s⁻¹ photosynthetic photon flux density (PPFD), 14-h photoperiod, and at a temperature of 27±2°C. The type of explant markedly influenced organogenesis and growth of the regenerated shoots. The regeneration frequencies were higher with leaf explants, while the number of shoots formed per responsive explant was greater with internode explants. However, the growth of regenerated shoots from internodes was seriously affected by vitrification. The number of shoots produced per responsive leaf explant increased from one to seven as the percentage of leaf explants producing shoots increased from 20 to more than 80%. NAA at 0.05 μM in combination with BA at 0.5μM induced the highest regeneration rate (87±8.8%) after 20 d of culture, yielding 5.9±0.8 shoots per responsive leaf explant. Histological examination confirmed the occurrence of direct organogenesis. The regenerated shoots from the best induction treatment were transferred to a fresh medium without plant growth regulators for 30 d. Finally, the elongated shoots were rooted by pre-treatment in an aqueous solution of NAA at 500 μM for 2 h and transferred to 1/4 MS. All plantlets raised in vitro were phenotypically normal and successfully hardened to ex vitro conditions. An experimental field plot with 2-yr-old in vitro-regenerated plants was established.     

High-frequency shoot regeneration from cotyledon explants of watermelon cv. sugar baby

Summary A protocol for in vitro shoot regeneration from cotyledon explants of Citrullus lanatus (Thunb.) Matsum. & Nakai cv. Sugar Baby is described. The cotyledons excised from 7-d-old aseptic seedlings showed the highest percentage of shoots on Murashige and Skoog (MS) + N⁶-benzyladenine (BA; 3.0 μM) + N⁶-[2-isopentenyl] adenine (2iP; 3.0 μM) and MS + BA (3.0 μM) + indole-3-acetic acid (IAA; 3.0 μM). Whereas the latter medium induced shoot regeneration after the callusing of the explant, the former stimulated direct shoot formation. The regenerated shoots were rooted and the resulting plants were established in earthen pots with 55% success.     

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.     

In vitro propagation of limonium wrightii (Hance) Ktze. (Plumbaginaceae), an ethnomedicinal plant, from shoot-tip, leaf- and inflorescence-node explants

Summary Rapid in vitro propagation of Limonium wrightii (Hance) Ktze. (Plumbaginaceae), an endangered medicinal plant, was achieved by culturing the shoot-tip (primary and lateral), leaf- and influorescence-node explants. MS (Murashige and Skoog, 1962) medium supplemented with 8.87 μMN⁶-benyladenine (BA) and 1.07 μM α-naphthaleneacetic acid (NAA) supported induction of adventitious shoots from the shoot-tip, inflorescence-node and middle and basal parts of leaf explants after 60 d of culture. Adventitious shoots were multiplied by subculturing on MS medium supplemented with BA (2,21–17.75 μM) in combination with NAA (1.07 μM). The percentage of explants forming shoots and the average number of adventitious shoot buds produced per explant were stimulated by increasing the strength (1/4x, 1/2x, 1x, 2x) of the MS medium. Shoots were rooted on MS basal medium with 4.92 μM indole-3-butyric acid. Plantlets with a morphologically normal appearance produced from adventitious shoots were transferred to soil and acclimated in the growth chamber for 1 mo.     

Micropropagation of chinese redbud (<Emphasis Type="Italic">Cercis yunnanensis</Emphasis>) through axillary bud breaking and induction of adventitious shoots from leaf pieces

Summary Factors affecting in vitro shoot production and regeneration of Cercis yunnanensis Hu et Cheng were investigated by comparing various growth regulators and explant types. For optimum shoot production from axillary buds, Murashige and Skoog (MS) media containing 6-benzyladenine, either alone or in combination with a low concentration of thidiazuron, resulted in the greatest number of shoots formed per explant (>3). Explants (2 mm long) containing one axillary bud placed in directcontact with the medium yielded the most shoots per bud (1.6) when grown on growth regulator-free medium. Root formation on 70–80% of shoot explants was accomplished using either indole-3-butyric acid or α-naphthaleneacetic acid in the medium, with significantly more roots formed on explants possessing and apical bud than those without the bud. Direct shoot organogenesis from leaf explants occurred on MS medium containing 10–30 μM thidiazuron, with up to 42% of leaf explants producing shoots.     

High-frequency plant regeneration from cotyledon callus of <Emphasis Type="Italic">Acacia sinuata</Emphasis> (Lour.) Merr

Summary A new reliable protocol for the induction of adventitious shoots and plant regenertion from cotyledon-derived callus of Acacia sinuata has been developed. Calluses were induced from cotyledon explants on Murashige and Skoog (MS) medium containing 3% sucrose, 0.8% agar or 0.15% phytagel, 8.1 μM α-naphthaleneacetic acid, and 2.2 μM 6-benzylaminopurine (BA). High-frequency regeneration of adventitious buds from callus was achieved when cultured on half-strength MS medium supplemented with 10% coconut water, 13.3 μM BA, and 2.5 μM zeatin. Histological studies revealed that the regenerated shoots originated from the callus. Among the various carbohydrates tested, sucrose at 87.6 mM was optimum for shoot-bud induction. Addition of 1.7 μM gibberellic acid along with 4.4 μM favored shoot elongation. In vitro-raised shoots produced prominent roots when transferred to half-strength MS medium supplemented with 7.4 μM indole-3-butyric acid. Rooted plants, thus developed, were hardened and successfully established in soil (45%). This protocol yielded an average of 40 plantlets per cotyledon explant over a period of 3 mo.     

Adventitious bud regeneration from leaf and cotyledon explants of Chinese hawthorn (<Emphasis Type="Italic">Crataegus pinnatifida</Emphasis> Bge. var. <Emphasis Type="Italic">major</Emphasis> N.E.Br.)

Hawthorn (Crataegus spp.) is an important plant with a long history as an ornamental and a source of medicine. A protocol is outlined for adventitious bud regeneration from leaf and cotyledon explants of Chinese hawthorn (C. pinnatifida Bge. var. major N.E.Br.). Adventitious buds were induced on both the leaves of sprouting winter buds and the leaves of in vitro plants, but the percentage of bud regeneration from leaves of in vitro plants was very low—less than 6%. On N6 medium supplemented with 31.08 μM BA and 9.67 μM NAA, the percentages of bud regeneration from leaves of sprouting winter buds of cultivars “Liaohong” and “Qiujinxing” were 31.4% and 17.6%, respectively. The regeneration abilities of three kinds of cotyledon explants, immature cotyledon, mature cotyledon, and cotyledon leaf, were compared. The percentage of bud regeneration from cotyledon leaves was higher. On MS media supplemented with 4.44 μM BA and 4.54–9.08 μM TDZ, the percentages of bud regeneration from cotyledon leaves of cultivars “Qiujinxing” and “Xiajinxing” were 27.7 ± 7.8% and 20.1 ± 4.7%, respectively, and the numbers of buds per explant were 5.9 ± 1.6 and 3.2 ± 0.7, respectively. On B5 medium supplemented with 2.22 μM BA, 2.32 μM Kn, and 0.57 μM IAA, adventitious buds grew quickly and 80–100% of buds developed into shoots. The shoots rooted successfully with the two-step rooting method. Ninety days after transplantation, more than 80% plants were survived. This system of adventitious bud regeneration from leaf and cotyledon explants could be useful for the genetic transformation and polyploidization of Chinese hawthorn.     

Strawberry sepal: Another explant for thidiazuron-induced adventitious shoot regeneration

Summary An efficient system to regenerate shoots on excised sepals (calyx) of greenhouse-grown ‘Bounty’ strawberry (Fragaria x ananassa Duch.) was developed in vitro. Sepal cultures produced multiple buds and shoots without an intermediary callus phase on 2–4 μM 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea (thidiazuron, TDZ)-containing shoot induction medium within 4–5 wk of culture initiation. Young expanding sepals with the adaxial side touching the culture medium and maintained for 14 d in darkness produced the best results. In a second experiment, sepals proved more effective than the leaf discs and petiole segments for regenerating shoots. A third experiment compared the effects of six concentrations of two cytokinins (TDZ at 0, 0.5, 2, and 4 μM and zeatin at 2 and 4 μM) for elongation of sepal-derived adventitious shoots. The media containing TDZ generally promoted more callus formation and suppressed shoot elongation. TDZ-initiated cultures transferred into the medium containing 2–4 μM zeatin, produced usable shoots after one additional subculture. Shoots were rooted in vitro in the same medium used for shoot regeneration, but without any growth regulators. When transferred to potting medium, 85–90% of in vitro plantlets survived.     

Efficient Plant Regeneration System From Leaf Discs of Zonal (Pelargonium x hortorum) and two scented (P. capitatum and P. graveolens) Geraniums

An efficient and rapid plant regeneration system was established for zonal and scented geraniums using leaf discs as explants. Several explants, medium and culture conditions were studied to optimize shoot induction. Leaf discs taken from 4–5 weeks old in vitro grown plants, whatever the genotype, were more effective for shoot regeneration than those taken from greenhouse grown plants. Darkness proved to be a stimulating factor for shoot regeneration and the combination between NAA and two cytokinins gave the best results. Direct shoot regeneration (100%) was obtained from leaf discs of P. capitatum on half-strength MS medium supplemented with 0.5 mg l⁻¹ NAA in combination with 1 mg l⁻¹ of BAP and zeatin in darkness (11.4 shoots per explant). In the same medium and culture conditions, all P. graveolens leaf discs also exhibited direct shoot regeneration (7.3 shoots per explant). For P. x hortorum, 100% of leaf discs underwent shoot regeneration on a MS medium supplemented with 0.2 mg l⁻¹ NAA in combination with 0.5 mg l⁻¹ of BAP and zeatin in darkness (8.8 shoots per explant) or under low light conditions with 0.2 mg l⁻¹ NAA and 1 mg l⁻¹ of BAP and zeatin (7.5 shoots per explant). For this species, the best results for shoot elongation were obtained on half-strength MS medium gelled with Phytagel 0.3% (v/v). Whatever the genotype, all shoots rooted readily when transferred to diluted MS medium (MS/2) containing 1 mg l⁻¹ IAA. Acclimatized plants grew normally and flowered in greenhouse conditions. Flow cytometry analysis made on leaves of acclimatized plants revealed that all the scented geranium plants are similar to mother plants while 71% of P. x hortorum plants which showed strong growth were tetraploid.     

Efficient and simple plant regeneration via organogenesis from leaf segment cultures of persimmon (Diospyros kaki thunb.)

Summary An efficient and simple plant regeneration system via organogenesis from leaf segments of persimmon (Diospyros kaki Thunb.) cultivars ‘Fuyu’ and ‘Nishimurawase’ has been developed. The regeneration capacity was influenced by the culture vessels, gelling agents, plant growth regulators, and light conditions. Leaf explants taken from in vitro shoots were cultured on a modified Murashige and Skoog medium (MS1/2N), for 16 wk without transfer to fresh medium. Adventious shoots appeared after 4 and 8 wk in culture of ‘Nishimurawase’ and ‘Fuyu’ tissues, respectively. The culture of leaf explants in Erlenmeyer flasks with medium containing 4 g l⁻¹ agar enhanced shoot formation in comparison to media with increased agar concentrations. Optimal shoot regeneration was obtained with 5 mg l⁻¹ (22.8 μM) zeatin and 0.1 mg l⁻¹ (0.05 μM) indole-3-butyric acid (IBA) for ‘Nishimurawase’, and 10 mg l⁻¹ (45.6 μM) zeatin and 0.1 mg l⁻¹ (0.05 μM) IBA for ‘Fuyn’. Shoot regeneration frequencies in both cultivars were 100%, and shoot numbers per explant reached up to 9.2 for ‘Nishimurawase’ and 2.2 for ‘Fuyu’. Dark incubation during the first 4–5 wk was the most effective condition to successfully influence shoot regeneration in both cultivars. While dark incubation was essential for adventitious shoot formation by ‘Fuyu’, it was only slightly beneficial to ‘Nishimurawase’. More than 80% of the regenerated shoots rooted within 4 wk on hormone-free MS1/2N demium after having been dipped for 30 s in 250 mg l⁻¹ (1.1. mM) IBA solution.