Direct shoot bud induction and plant regeneration in <Emphasis Type="Italic">Capsicum frutescens</Emphasis> Mill.: influence of polyamines and polarity

Direct shoot bud induction and plant regeneration was achieved in Capsicum frutescens var. KTOC. Aseptically grown seedling explants devoid of roots, apical meristem and cotyledons were inoculated in an inverted position in medium comprising of Murashige and Skoog (Physiol Plant 15:472–497, 1962) basal medium supplemented with 2-(N-morpholine) ethanesulphonic acid buffer along with 2.28 μM indole-3-acetic acid, 10 μM silver nitrate and either of 13.31–89.77 μM benzyl adenine (BA), 9.29–23.23 μM kinetin, 0.91–9.12 μM zeatin, 2.46–9.84 μM 2-isopentenyl adenine. Profuse shoot bud induction was observed only in explants grown on a media supplemented with BA (26.63 μM) as a cytokinin source and 19.4 ± 4.2 shoot buds per explant was obtained in inverted mode under continuous light. Incorporation of polyamine inhibitors in the culture medium completely inhibited shoothoot bud induction. Incorporation of exogenous polyamines improved the induction of shoot buds under 24 h photoperiod. These buds were elongated in MS medium containing 2.8 μM gibberellic acid. Transfer of these shoots to hormone-free MS medium resulted in rooting and rooted plants were transferred to fields. This protocol can be efficiently used for mass propagation and presumably also for regeneration of genetically transformed C. frutescens.     

Influence of preculture treatment and types of explants on shoot growth and in vitro flowering of feathered amaranth (Celosia argentea var. plumose)

The shoots developed from both the shoot tip and nodal explants of feathered amaranth (Celosia argentea var. plumosa—feathered cockscomb or plumed cockscomb) after 8 weeks of culture in the presence of either paclobutrazol or benzyladenine (BA) were shorter than those developed on basal Murashige and Skoog (MS) medium (Physiol Plant, 15:473–497, 1962) alone. However, this retarding effect was more pronounced in the nodal explant culture. Shoot tip explants from 2-week-old seedlings were more adversely affected by 0.85 or 1.7 μM paclobutrazol than those from older seedlings. In contrast, regardless of preculture duration investigated nodal explants did not exhibit different response to three different concentrations of paclobutazol. The response to 2.2 or 4.4 μM BA appeared to be largely independent of the age of the shoot tip explants or preculture treatment of nodal explants. Shoots developed from nodal explants produced a higher number of terminal inflorescence than those from shoot tip explants. Moreover, only lateral shoots from nodal explant culture formed inflorescence. Increased preculture duration on basal MS medium could generally lessen the inhibitory effect of lower concentrations of paclobutazol or BA on terminal or lateral inflorescence formation in nodal explant culture.     

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.     

Plant Regeneration from Immature Embryo Cultures of Vigna unguiculata

Micropropagated plants of two annual haloxerophytic Asiatic Salsola species (S. pestifer and S. lanata) were obtained from zygotic embryos cultured on Murashige and Skoog (MS) agar medium supplemented with 0.5 μM benzylamino-purine (BAP) and 0.3 μM indole-3-acetic acid (IAA) or with 0.5 μM 6 γ, γ-dimethylallylaminopurine and 0.3 μM IAA. The callus induction from shoot and leaf explants derived from plants propagated in vitro were obtained on MS agar medium with various concentration of auxins and cytokinins. The best medium for growth and proliferation of calluses of both studied species was MS medium containing 9.0 μM 2,4-dichlorophenoxyacetic acid. It was also determined that beginning of plant regeneration from callus of S. lanata was induced by 8.8 μM BAP. This revised version was published online in July 2006 with corrections to the Cover Date.     

In vitro regeneration of sesame (Sesamum indicum L.) from seedling cotyledon and hypocotyl explants

The goal of this study was to develop an efficient regeneration protocol to be used for genetic transformation of sesame. Published regeneration methods using benzyladenine (BA) and 1-naphthalene acetic acid (NAA) were unsuccessful for the cultivars used herein. Experiments were carried out using cotyledon and hypocotyl explants from the cultivar Mtwara-2. Later the optimised culture conditions were used to investigate the regeneration response of different genotypes. There was significant interaction between hormone treatments and macronutrients for shoot and root regeneration. Results also showed that shoot regeneration was significantly influenced by explant type. Shoots were only obtained from cotyledons whereas both cotyledons and hypocotyls could produce roots. Modified Murashige and Skoog (MS) medium with N6 macronutrients resulted in twice the shoot regeneration frequency obtained with ½MS macronutrients in the presence of thidiazuron (TDZ). The shoot regeneration frequency was significantly reduced when BA was used in place of TDZ. On shoot regeneration medium containing BA and NAA, only roots were formed. Replacing NAA with indole-3-acetic acid (IAA) greatly improved the regeneration of shoots. The optimum growth regulator combination for shoot regeneration was 20 μM TDZ together with 2.5 μM IAA, which gave a frequency of 63% and 4.4 shoots per regenerating explant for the best cultivar Ex-El. Genotypic differences were significant both for the number of explants regenerating shoots and the number of shoots produced per regenerating explant.     

An improved micropropagation of <Emphasis Type="Italic">Spilanthes acmella</Emphasis> L. through transverse thin cell layer culture

An efficient and improved in vitro propagation system for Spilanthes acmella L. using transverse thin cell layer (tTCL) culture system was established. The frequency of shoot regeneration from tTCL nodal segments was affected by concentrations of plant growth regulators and orientation of the explant. MS (Murashige and Skoog in Physiol Plant 15:473–497, 1962) medium with 5.0 mg dm⁻³ BAP was optimal for shoot regeneration. Upon this medium, the explant inoculated in the upright orientation exhibited a high frequency of shoot regeneration (about 97%), and the highest number of shoots (31.5) per explant. The intact node (1.0–1.5 cm) cultured on the same medium had significantly lower shoot multiplication ability with only 4.5 shoots per responsive explant. As compared to BAP alone, the combination of BAP and Kin or NAA did not have positive effects on shoot multiplication from tTCL nodal segments. Rooting of shoots was achieved on growth regulator free full-strength MS medium. Plantlets were transplanted into soil with 90–100% survival rate.     

Influence of different ethylene inhibitors on somatic embryogenesis and secondary embryogenesis from <Emphasis Type="Italic">Coffea canephora</Emphasis> P ex Fr.

The effect of cobalt chloride, salicylic acid, and silver nitrate for embryogenesis was studied in in vitro cultures of Coffea canephora. Murashige and Skoog (in Physiol. Plant. 15:473–497, 1962) medium containing 20 and 40 μM either of cobalt chloride, silver nitrate, or salicylic acid supplemented with 1.1 μM N ⁶ benzyladenine and 2.85 μM indole-3-acetic acid was used for the study. At 20 and 40 μM silver nitrate treatment, 35–48% explants responded for embryogenesis, and 38 ± 7 and 153 ± 27 embryos were produced from each callus mass, respectively, whereas only 5% control explants responded on medium devoid of silver nitrate, cobalt chloride, or salicylic acid. Secondary embryogenesis was observed in 70–90% of the explants, and around 100–150 embryos were produced from each explant cultured on a medium containing silver nitrate, and only a 3% response was noticed in control embryo explants. Yellow friable embryogenic calluses were obtained from the cut edges of most of the tissues grown in a medium supplemented with cobalt chloride. The results clearly demonstrated that, among the tested ethylene inhibitors, silver nitrate is very effective in reprogramming the cellular machinery toward embryogenesis.     

High-frequency plant regeneration via adventitious shoot formation from deembryonated cotyledon explants of Sesamum indicum L.

Sesamum indicum L. was used as an important oil crop in the world. An efficient protocol for in vitro plant regeneration via adventitious shoot formation from deembryonated cotyledon explants isolated from mature seeds of sesame is developed. Optimal medium for direct adventitious shoot formation was Murashige and Skoog (MS) medium with 22.2 μM 6-benzylaminopurin (BA) and 5.7 μM indole-3-acetic acid (IAA). Abscisic acid (3.8 μM ABA) and AgNO₃ (29.4 μM) were effective in enhancing the frequency of adventitious shoot formation. Preculture of cotyledon explants on high sucrose concentration (6–9%) for 2 wk and subsequent transfer to 3% sucrose enhanced the frequency of adventitious shoot induction. Root formation from the adventitious shoots was easily achieved on MS medium containing 2.7 μM of α-naphthalene acetic acid (NAA). Regenerated plantlets were acclimatized on sand and peat moss (1:1), showing 95% survival with subsequent flowering and seed set. We established the high-frequency plant regeneration via adventitious shoot formation in S. indicum L.     

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.     

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.     

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.     

Factors influencing direct shoot regeneration from ovary explants of saffron

Direct adventitious shoot regeneration from ovary explants of Crocus sativus L. was influenced by media components, incubation conditions, and age of the explant. The best response towards caulogenesis (28%) with highest shoot numbers per ovary was observed when full strength Murashige and Skoog (1962) medium was supplemented with naphthaleneacetic acid and benzyladenine. Incubation in the dark at 20 °C was beneficial for induction of shoot buds. Ovaries of different growth stages having stigmas of pale yellow, pale orange and bright orange regenerated a maximum mean number (3.8 – 4.2) of shoots per ovary. Further development of ovary-derived shoots was influenced by the composition of basal salts in the culture medium where full strength Murashige and Skoog salts gave the best response of those tested. Regenerated shoots produced normal photosynthetic leaves and corms. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

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     

Factors influencing efficiency of shoot regeneration in <Emphasis Type="Italic">Ziziphus jujuba</Emphasis> Mill. ‘Huizao’

Several factors affecting the frequency of leaf regeneration of Ziziphus jujuba ‘Huizao’ were investigated in this study, including basal medium, plant growth regulators, leaf maturity, explant orientation, and additive chemicals. The results showed that the highest shoot regeneration frequency (82.25%) was obtained on woody plant medium supplemented with 2.27 μM thidiazuron, 1.07 μM α-naphthalene-acetic acid (NAA) and 2.94 μM silver nitrate after a 10 days incubation in darkness. This study also suggested an increased regeneration frequency of expanded young leaves which were taken from the mid-stem position, as well as from the explant abaxial surface contacting with the medium. In addition, hyperhydric of adventitious buds could be effectively reduced by adding 40 g l⁻¹ sucrose to the medium. A 95.56% rooting rate could be produced from shoots cultured on half-strength Murashige and Skoog (in Physiol Plant 15:473–497, 1962) medium plus 2.69 μM NAA.     

<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.     

Pepper (<Emphasis Type="Italic">capsicum annuum</Emphasis> L.) regenerants obtained by direct somatic embryogenesis fail to develop a shoot

Summary Three auxin-type herbicides, namely 2.4-dichlorophenoxyacetic acid (2,4-D), (4-chlorophenoxy)acetic acid 2-(dimethylamino)ethyl ester (centrophenoxine), and quinolinecarboxylic acid (quinclorac) induced direct somatic embryogenesis in seed-derived zygotic embryo explants of sweet pepper (Capsicum annuum L.) when added to Murashige and Skoog medium with 200 mM sucrose. Optimum concentrations for embryogenesis induction were 0.40–0.45 mM and 1.15–1.30 μM for 2.4-D and centrophenoxine, respectively (in the presence of 5.0 gl⁻¹ activated charcoal), or 40 μM for quinclorac (in medium without activated charcoal). Somatic embryos emerged from the epidermal and subepidermal tissues and developed on the surface of the explant. Centrophenoxine- or 2.4-D-mediated embryogenesis was accomplished from 95% of the explants in about 3 wk and, on average, six embryos were formed per explant. Induction efficieney was lower for quinelorac. Centrophenoxine-mediated embryognesis was possible in 10 pepper cultivars, the extent of the reponse-being genotype-dependent. embryos detached from the explant and transplanted onto a growth regulator-free medium germinated; however, the recovered regenerants were without a shoot, and some of them bore a single deformed cotyledon while others had no cotyledons. Regenerants lacking a shoot were generated irrespective of the auxin type applied and across all responsive genotypes investigated. Absence of a shoot, resulting from a failure in the establishment of a normal functioning apical shoot meristem, was the principal developmental disorder that precluded regeneration of normal plants via direct somatic embryogenesis. Since stem cells of the shoot meristem become established in globular and heart-stage embryos, we deduce that the absence of a shoot in germinating embryos could orginate from deviant differentiation at these early stages of embryogeny.     

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.     

<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.     

Auxin pretreatment promotes regeneration of sugarcane (Saccharum spp. hybrids) midrib segment explants

We have developed a new, simple, quick and genotype-independent method for direct regeneration of sugarcane using novel midrib segment explants. Our protocol involves two steps: the pretreatment of starting material on MS (Murashige and Skoog (1962) Physiol Plant 15:473–497) medium containing 3.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) for 8 days under continuous dark and subsequent transfer of the explants to MS medium augmented with 0.1 mg/l benzyladenine (BA) and 0.1 mg/l naphthaleneacetic acid (NAA) under light-dark conditions. On the regeneration medium, numerous globular structures appeared from the explants and subsequently differentiated into shoots. Regenerated shoots attained 2–5 cm height within 30 days of culture initiation and readily rooted on MS basal medium. Hardened plants were successfully established in the greenhouse. The regulation of sugarcane morphogenesis by auxin pretreatment is discussed.