Micropropagation of Cowania subintegra and Cowania stansburiana

Both Cowania subintegra Kearney and C. stansburiana Torr. were successfully propagated in vitro. Shoot proliferation occurred from shoot tips of green-house grown C. subintegra using a modified Murashige and Skoog medium supplemented with 4.4 M 6-benzyladenine and 0.5 M indole butyric acid. Excised microshoots (1.5–3.0 cm long) of both species were rooted using a two-step process in which they were cultured for 3 days in a root initiation medium with 2.7 M naphthaleneacetic acid and then transferred to a low nitrogen root elongation medium without auxin. Plantlets were successfully transferred to soilless potting mix.     

Micropropagation of Mexican redbud,Cercis canadensis var. mexicana

Mexican redbud (Cercis canadensis var. mexicana) shoot cultures were initiated from explants taken from both mature and juvenile stock plants. Culture conditions affecting shoot growth and proliferation and rooting of three clones were investigated. Shoot growth was best on media supplemented with 0.25% activated charcoal and solidified with 0.2% Gelrite. Four commercially available salt formulations (Anderson's rhododendron medium, WPM, MS, DKW) were tested for growth of shoot cultures, and Anderson's rhododendron basal salt mixture was superior. Axillary shoots grew from explants cultured media supplemented with a wide range of concentrations of benzyladenine and thidiazuron. Benzyladenine at 5.6–22.2 M supported the best combination of shoot quality and number. Rooting of microshoots in vitro was best on half-strength WPM containing 6.71 M naphthaleneacetic acid and 0.1% activated charcoal.Abbreviations BA 6-benzyladenine - IBA indole-3-butyric acid - 2iP 6-(, -dimethylallylamino)purine - DKW Driver & Kuniyuki Walnut - kinetin 6-furfurlaminopurine - MS Murashige & Skoog - NAA -naphthaleneacetic acid - WPM Woody Plant Medium - TDZ thidiazuron - 1-phenyl-3 (1,2,3-thidiazol-5-yl)urea     

Micropropagation of Raphanus sativus L. var. longipinnatus (Japanese radish) cv. Gungjung

Shoot cultures were established from seedling shoot tips of Raphanus sativus var. longipinnatus Bailey cv. Gungjung, (Japanese radish) cultured on a Murashige-Skoog medium supplemented with ca. 4.5–135 M kinetin or N⁶-benzyladenine. The latter cytokinin supported overall better growth, and 22.2 M was adopted for maintenance of established cultures. The nitrate: ammonium levels in the medium proved optimal for growth and shoot proliferation and both these parameters were significantly increased by addition of adenine sulfate or sodium phosphate. Rooting of excised shoots was achieved on auxin containing medium. Indole-3-butyric acid (ca. 5 or 10 M) also enhanced shoot growth. Plants were easily established in soil, appeared morphologically normal, and flowered.     

Shoot regeneration and cryopreservation of shoot tips of apple (Malus) by encapsulation–dehydration

Here, we report an efficient and widely applicable method for cryopreservation of Malus shoot tips by encapsulation–dehydration using adventitious shoots. Shoots were induced from leaf segments cultured on a shoot induction medium containing 2–3 mg L^?1 thidiazuron, depending on genotype, and 0.5 mg L^?1 indole-3-butyric acid. Shoot tips (3 mm in length) containing six leaf primordia excised from 11-wk-old adventitious shoots were encapsulated and precultured with 0.5 M sucrose for 5 d, followed by air-drying for 6 h prior to direct immersion in liquid nitrogen. With our protocol, we obtained a mean organogenesis rate of 100%, a mean of 4.5 adventitious shoots per explant (leaf segment), and a mean shoot recovery of 57.0% from cryopreserved shoot tips in four Malus species. Inter-simple sequence repeat (ISSR) analysis did not reveal any polymorphic bands in regenerants recovered from either leaf segments or cryopreserved shoot tips of ‘Gala’. To the best of our knowledge, this is the first report on cryopreservation of Malus shoot tips using adventitious shoots derived from leaf segments and is the most widely applicable protocol so far reported for cryopreservation of Malus. Establishment of this protocol provides an alternative means for cryopreservation of Malus.     

The effect of in vitro pretreatments on subsequent shoot organogenesis from excised Rubus and Malus leaves

Shoot regeneration from Rubus leaves was obtained on a medium containing MS salts, vitamins and sugars, Staba vitamins, casein hydrolysate (100 mg l^–1) and 10 M thidiazuron. Shoot regeneration from Malus leaves was obtained on N⁶ rice anther medium with 5 M thidiazuron. In vitro pretreatment of source shoots with either colchicine or thidiazuron enhanced the organogenic potential of detached leaves of two Rubus hybrids. The response to colchicine was quadratic and occurred at non-mutagenic concentrations (75–250 M). The response to thidiazuron was exponential between 0 and 5 M. When applied as a pretreatment, the effectiveness of several different cytokinins (benzyladenine, thidiazuron, zeatin) at enhancing Malus and Rubus organogenesis was related to the shoot proliferation activity of the cytokinin and to treatment-induced variation in leaf and petiole size.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indolebutyric acid - MS Murashige & Skoog basal medium devoid of plant growth regulators - OI organogenesis-initiating subculture - PTI colchicine pretreatment subculture - PTII cytokinin pretreatment subculture - NAA naphthaleneacetic acid - TDZ thidiazuron - zeatin trans-zeatin     

In vitro propagation of prairie gentian

Explants of shoot tips, internodal stem sections, and leaf segments of Lisianthus, Eustoma grandiflorum (Griseb.) Schinners, Dwarf Purple were cultured in vitro on modified Murashige and Skoog (MS) media. Explants of shoot tips and internodal stem sections developed into multiple shoots, whereas, leaf segments turned chlorotic on a medium supplemented with 3 mgl^-1 benzyladenine (BA) and 0.2 mgl^-1 naphthalene acetic acid (NAA). Shoot proliferation was obtained on shoot tips and leaf segments with 3 mgl^-1 BA, but internodal stem sections became necrotic and died on this medium. Rooting was induced in cultures with multiple shoots by subculturing explants on a half-strength MS medium supplemented with 2 mgl^-1 indole-3-acetic acid (IAA). Rooted plantlets were successfully transferred to soil.     

The cryopreservation of shoot tips of Rosa multiflora

Shoot tips of in vitro grown plantlets of Rosa multiflora were cryopreserved using an encapsulation/dehydration procedure. The influence of sucrose and silica gel pretreatments on pre- and post-freeze shoot growth were examined. Shoot tips recovered from liquid nitrogen only grew after 24h pretreatment in medium containing 0.5 M sucrose, followed by 2 h drying with silica gel and rapid freezing.Abbreviations RSC1 modified Murashige and Skoog medium for Rosa multiflora shoot culture     

Micropropagation for recovery of Cucumis hystrix

A micropropagation protocol that allows for the efficient cloning of C. hystrix was developed using 1 mm shoot-tip explants prepared from plants grown in the greenhouse. Establishment of Stage I cultures was greatest (83%) when shoot tips were cultured in modified MS (Murashige and Skoog, 1962) medium containing (per liter) 30 g sucrose, 0.1 g myo-inositol, and 5 g Agargel plus 1.7 M indole-3-butyric acid (IBA), 0.5 M kinetin and 0.3 M gibberillic acid (GA₃) (IKG). Benzyladenine (BA, 5 M) proved best for Stage II shoot proliferation. Over 35 shoots were obtained per vessel (five shoots per vessel) when explants were cultured in medium containing BA. Less than 10 shoots were obtained per vessel when kinetin (0.5–5 M) and IKG were used. Stage II cultures established from 1 cm shoot tips obtained from Stage I shoots produced more shoots (1.3-fold) than single node explants. Rooting and plantlet acclimatization were best when shoots greater than 1.5 cm were incubated in MS medium containing 0.5 M naphthalene acetic acid (NAA). Higher NAA concentrations stimulated rooting but inhibited shoot elongation and reduced the ability of plantlets to survive acclimatization to ambient conditions. Plantlet height influenced acclimatization. Over 72% of plantlets survived acclimatization if they were at least 4.6 cm when transferred to soilless growing medium.     

A highly efficient in vitro regeneration methodology for mature Chinese tallow tree (Sapium sebiferum Roxb.)

A highly efficientin vitro regeneration methodology for mature chutese tallow tree (Sapium sebiferum Roxb.) has been developed. Shoot segments cultured on MS medium supplemented with 7.5 µM NAA produced light green callus. Optimum shoot differentiation resulted when callus was transferred to MS medium with 1 µM BA and 0.25 µM NAA. Shoot forming ability of callus was higher on MS medium compared to B5, half-MS or WPM. A continuous shoot harvest system at four-week intervals was established. Shoot yield continued for six months without loss of vigour. Regenerated shoots were rooted by culturing on half strength agar-gelled MS medium containing 1 µM IBA. Rooted plantlets were transferred to 1:1 soil vermiculite mixture and acclimatized with 67 % survival rate. Fully acclimatized plants were planted in the field, and performance is being evaluated.Abbreviations 2, 4- D 2, 4-dichlorophenoxyacetic acid - NAA 1 - napthaleneacetic acid - BA benzyladenine - Kn kinetin - 2-ip 6 - (, -dimethylallylamino)-purine - IBA indole-3-butyric acid - WPM woody plant medium (1980) - MS Murashige and Skoog (1962) medium - B5 Gamborg et al's medium (1968)     

Plant regeneration from various expiants of cultivated Piper species

Morphogenetic potential of root, leaf, node and internode expiants of 3 cultivated Piper species was investigated to develop a reliable plant regeneration protocol. P. longum (pipli) was the most responsive followed by P. betle (betel vine) and P. nigrum (black pepper). In P. longum the highest number of shoot buds was produced on root expiants followed by node, internode and leaf expiants. In P. betle and P. nigrum adventitious shoot buds differentiated only from internodal and nodal ring regions, respectively. Histological examination in P. longum showed that adventitious shoot buds originate directly from the cortical cells of the root and the internode without an intervening callus phase. Benzyladenine was superior to kinetin for shoot induction and its optimum concentrations for P. longum, P. betle and P. nigrum were 1–2, 10 and 10 M, respectively. Shoot elongation and rooting were achieved in B₅ medium containing 0.5 M benzyladenine and 1 M indoleacetic acid, respectively. Regenerated plants were established in soil.Abbreviations BA N⁶ benzyladenine - B₅ medium Gamborg et al. (1968) medium - IAA indole-3-acetic acid     

Micropropagation of a recalcitrant male asparagus clone (MD 22-8)

TheAsparagus officinalis male asparagus clone MD 22–8 (Howard Scott) is highly prized by asparagus breeders but has been very difficult to micropropagate due to its slow growth rate and reluctance to initiate roots. Shoot tips cultured on indole-3-acetic acid or indole-3-propionic acid, at concentrations of 1.53 and 1.44 M respectively, resulted in levels of root initiation and elongation equal to or better than those of a female asparagus clone. These culture conditions also improved the rates of shoot initiation and elongation. Root initiation and elongation were further increased by culture conditions that provided enhanced gas exchange.Abbreviations BA 6-benzyladenine - IAA 3-indoleacetic acid - IPA indolepropionic acid - NAA -naphthaleneacetic acid - 2,4-d 2,4-dichlorophenoxyacetic acid - 2,4,5-T 2,4,5-trichlorophenox-yacetic acid - PPF photosynthetic photon flux - MS Murashige & Skoog     

Rapid in vitro adventitious shoot propagation of <Emphasis Type="Italic">Scopolia parviflora</Emphasis> through rhizome cultures for enhanced production of tropane alkaloids

A rapid micropropagation system for Scopolia parviflora Nakai (Solanaceae), a rare medicinal plant native to Korea, was established using rhizome cultures. Shoots that originated from adventitious shoots of the rhizome were multiplied when the rhizomes were cultured on half-strength B5 liquid medium supplemented with various growth regulators. Optimum shoot multiplication was observed in half-strength B5 medium containing 3% (w/v) sucrose and 5.77 M gibberellic acid (GA₃). Each rhizome gave rise to an average of 12 shoots. Shoot elongation and root induction from multiple shoots occurred on growth regulator-free half-strength B5 solid medium. Healthy plantlets were transferred to a peat moss:vermiculite mixture for acclimatization, which was successful. The concentrations of tropane alkaloids, hyoscyamine and scopolamine were determined in different tissues of native growing plants, in vitro-propagated plants and acclimatized plants by high-performance liquid chromatography. The analysis revealed that the levels of hyoscyamine and scopolamine were higher in in vitro-propagated plants than in the native growing plants. When the rhizome was cut into segments and transferred to optimal culture conditions for multiple shoot propagation, only 12 weeks were required to produce a mature plant. We conclude that in vitro propagation techniques through rhizome cultures provide an efficient and rapid method for shoot propagation of S. parviflora.Abbreviations BA Benzyladenine - 2,4-D 2,4-Dichlorophenoxyacetic acid - GA₃ Gibberellic acid - HPLC High-performance liquid chromatography - IBA Indole-3-butyric acid - NAA -Naphthaleneacetic acid     

Development of a common PVS2 vitrification method for cryopreservation of several fruit and vegetable crops

Many cryopreservation techniques are currently available, and it is common for new modifications to be developed for individual crops or specific genotypes. In this study, results of variations of the PVS2 cryopreservation protocol are compared to provide evidence for the suitability of a standard form of this technique for cryopreservation of a range of fruit, berry crops, and potato. Shoot cultures of Malus, Solanum, Lonicera, and Berberis were tested with variations of cold acclimation, pretreatment media, and PVS2 exposure times. A general protocol with some modifications was produced that was suitable for all four genera. The regenerative capacity of shoot tips after cryopreservation by this method exceeded a mean of 50% for Malus, Solanum, Lonicera, and Berberis, which is sufficient for setting storage in a cryobank. After liquid nitrogen storage, the shoot cultures that survived had a healthy appearance and developed rapidly. For each species tested, the only optimization required was the preparation of donor plants by cold acclimation and pretreatment. The choice of one common method simplifies the methodology for conducting experiments and storing a range of germplasm. The use of the PVS2 vitrification method with a 0.3-M sucrose pretreatment is multiuse and can be recommended as the most effective method for the cryopreservation of shoot tips from many plant species.     

Polyphenoloxidase-activity and -activation in embryogenic and non-embryogenic suspension cultures of Euphorbia pulcherrima

The activity and activation potential of polyphenoloxidase (PPO, E.C. 1.10.3.1.) of tissue from shoot tips, adult leaves and embryogenic and non-embryogenic cell suspension cultures of Euphorbia pulcherrima was investigated using an oxygen probe technique. PPO derived from differentiated in vivo plant tissue (shoot tips, leaves) cannot be activated either by storage at 0–4°, freezing and thawing, incubation with CaCl₂, sodium dodecyl sulfate or by incubation with trypsin. Embryogenic cells are characterized by high initial PPO activity and strong activation potential of membrane bound enzyme. Non-embryogenic material reveals low phenolase activity and low activation potential. An activation quotient (based on the ratio between PPO-activity determined after sodium dodecyl sulfate incubation to PPO-activity determined after CaCl₂-incubation) was calculated. This is independent of absolute enzyme activity and can be used for characterization of the embryogenic status of cells.Abbreviations fw fresh weight - MS Murashige and Skoog - PPO polyphenoloxidase - SDS sodium dodecyl sulfate     

In vitro culture of vanhoutte's spirea explants from ‘secondary cultures’ and dormant stems forced in solutions containing plant growth regulators

Explants from new growth of forced dormant stems and secondary cultures of Vanhoutte's spirea were cultured on Linsmaier and Skoog (L.S.) medium containing benzyladenine (BA), indoleacetic acid (IAA), thidiazuron (TDZ), and zeatin. The dormant stems were forced by immersing their basal portions in forcing solutions containing 626 µM 8-hydroxyquinoline citrate (8-HQC) and 2% sucrose. BA and gibberellic acid (GA₃) were also added into the forcing solutions to determine if explants obtained from the new growth will benefit from this treatment when culturedin vitro.L.S. medium supplemented with 5 µM BA alone, 5 µM BA plus 1 or 5 µM IAA, and 0.5 or 0.75 µM TDZ alone produced the best shoot proliferation for both sources of explants. BA and GA₃ appeared to be taken up from the forcing solution by the new softwood growth. BA in the forcing solution stimulatedin vitro shoot proliferation in different degrees depending on the period of treatment, while GA₃ caused lessin vitro shoot proliferation. It is proposed that forcing solutions containing plant growth regulators (P.G.R.) are a useful approach for manipulating responses of plant tissues culturedin vitro.     

In vitro propagation of Halesia carolina L. and the influence of explantation timing on initial shoot proliferation

Halesia carolina L., a small, ornamentally valuable tree, is difficult to propagate due to the complexity of seed propagation and the unavailability of propagules for conventional vegetative propagation. A micropropagation system was developed to facilitate easy propagation of this species. Actively growing shoot tips achieved optimum shoot proliferation from axillary buds when placed on Woody Plant Medium supplemented with 1.0 to 2.5 mg/l benzyladenine. The addition of 0.1 mg/l naphthaleneacetic acid had little effect on culture performance. Murashige and Skoog medium was incapable of supporting vigorous shoot proliferation. Non-sterile rooting conditions provided better rooting and subsequent plantlet growth, when compared to an in vitro rooting method. The seasonal fluctuations in the stock plant dramatically affected the shoot proliferating potential of the explants in vitro. Rapidly elongating shoots formed shoot proliferating cultures more slowly than explants taken either before or after the rapid elongation phase.     

In vitro propagation ofSalix tarraconensis Pau ex Font Quer,an endemic and threatened plant

Summary Salix tarraconensis Pau ex Font Quer, an endemic willow species from northeast Spain, was micropropagated with nodal segments. Shoot multiplication was obtained with different cytokinins, either on Murashige and Skoog medium or woody plant medium. Best results for shoot formation were obtained on Murashige and Skoog medium containing 4.9 μM of 6-γ-γ-dimethylallylaminopurine. Shoots showed strong apical dominance, and some cultures displayed apical necrosis. Benzyladenine gave the worst results; shoots displayed very slow growth, deformed leaves, and hyperhydrity. Good rooting of shoots was obtained with different auxins or without plant growth regulators on woody plant medium. The best results (90-100%) were obtained within 20 d. On rooting media with indole-3-butyric acid or indoleacetic acid, shoot elongation was good (35-40 mm length). Apical necrosis was observed in elongating shoots on rooting medium, but this disturbance favored axillary bud sprouting and formation of new shoots. Shoot length and quality of roots decreased gradually as the concentration of naphthaleneacetic acid increased. Plant survival was 90% 4 weeks after removal fromin vitro conditions.     

Roots as an enhancing factor for the production of artemisinin in shoot cultures of Artemisia annua

Artemisinin was produced in differentiated shoot cultures of Artemisia annua L. but was undetected in callus or cell cultures. The growth regulators benzyladenine, kinetin, chlormequat, and daminozide, at concentrations which severely reduced rooting, reduced artemisinin production. A highly significant correlation (1% level) was observed between shoot artemisinin content and number of roots (r=0.775^**), but shoot number and artemisinin content were unrelated (r=-0.198). Benzyladenine increased shoot proliferation at 0.5 and 5.0 M, but decreased root production at 0.5, 5.0, and 50 M. The highest levels of artemisinin production (0.287% DW) were obtained in hormone-free medium when root production was maximized. Removal of roots from shoots cultured in hormone-free liquid medium reduced shoot artemisinin by 53% and shoot arteannuin B by 60%. Neither artemisinin, arteannuin B, or artemisinic acid were detected from roots developed in semi-solid or liquid medium.Abbreviations BA benzyladenine - CCC chlormequat - DW dry weight - FW fresh weight - GA₃ gibberellic acid - GC/MS gas chromatography/mass spectrometry - HPLC-EC high-performance liquid chromatography with electrochemical detection - MS Murashige & Skoog basal medium - 2,4-d 2,4-dichlorophenoxyacetic acid Journal paper no. 14558 of Purdue Agricultural Research Progress     

Micropropagation of Ficus benjamina clones

In vitro culture of eight Ficus benjamina clones was initiated from shoot tips four times from January to June 1988. Shoot formation and growth in vitro were followed during eight subsequent subcultures, whereafter the developed shoot clusters were rooted in vitro. Significant differences among clones in proliferation rate and time to emergenece of first root in vitro were observed. A superior clone Cleo, previously selected for fast growth as a potted plant, also proved to have the highest proliferation rate and the shortest time until emergence of first root in vitro. The proliferation rate was nearly stabilized after five subcultures. A negative correlation between proliferation rate and time to emergence of first root in vitro was found.Abbreviations C clone - S subculture - SP(C) stock plant within clone - T time of initiation     

High-frequency induction of adventitious shoots from hypocotyl segments ofLiquidambar styracifiua L. by thidiazuron

The effects of thidiazuron (TDZ) on adventitious bud and shoot formation from hypocotyl segments of sweetgum (Liquidambar styracifiua) were tested alone and in combination with 2,4-dichlorophenoxyacetic acid (2,4-D). The combination of 1 mg/1 TDZ with 0.01 mg/l 2,4-D resulted in the highest frequency of bud production. Lower concentrations of TDZ stimulated shoot production, generating the most shoots at 0.1 mg/1 TDZ with 0.01 mg/1 of 2,4-D. Inhibition of shoot elongation by TDZ was overcome by transferring shoot cultures to a shoot proliferation medium lacking TDZ or containing naphthaleneacetic acid and benzyladenine in addition to TDZ. Shoot production in liquid culture was significantly greater than that in solid culture. Comparisons of in vitro and ex vitro rooting of the adventitious shoots demonstrated that ex vitro rooting produced plants with faster growth rates and more extensive root systems.Abbreviations BA Benzyladenine - IBA indole-3-butyric acid - NAA naphthaleneacetic acid - PGR plant growth regulator - TDZ thidiazuron - 2,4-D 2,4-dichlorophenoxyacetic acid