Establishment of Camptotheca acuminata regeneration from leaf explants

Plantlet regeneration through shoot formation from young leaf explant-derived callus of Camptotheca acuminata is described. Calli were obtained by placing leaf explants on Woody plant medium (WPM) supplemented with various concentrations of 6-benzyladenine (BA) and naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D). Callus induction was observed in all media evaluated. On the shoot induction medium, the callus induced on the WPM medium containing 19.8 μM BA and 5.8 μM NAA was the most effective, providing high shoot regeneration frequency (70.3 %) as well as the highest number of shoots (11.2 shoots explant⁻¹). The good rooting percentage and root quality (98 %, 5.9 roots shoot⁻¹) were achieved on WPM medium supplemented with 9.6 μM indole-3-butyric acid (IBA). 96 % of the in vitro rooted plantlets with well developed shoots and roots survived transfer to soil.     

Improvement of in vitro propagation of apricot cultivar ‘Bebecou’

Factors affecting successful establishment in vitro, rapid proliferation and rooting of apricot cultivar ‘Bebecou’ were studied. Ethanol and NaOCl were applied in several combinations for disinfection; chilling, plant growth regulators BA, IAA and GA₃, antibiotics, different culture vessels and systems of subculture were evaluated for the optimization of shoot proliferation and the auxins NAA and IBA were assessed for root induction. The highest number of new microshoots/explant (18.7) was obtained in a culture medium supplemented with 2.2 μM BA+0.57 μM IAA after 300 h of chilling. The effect of GA₃ (11.4 μM) on shoot proliferation was positive in combination with 4.4 or 8.9 μM BA. Shoot length and productivity were highest at 2.2 μM BA+11.4 μM GA₃+0.57 μM IAA and at 2.2 μM BA+0.57 μM IAA, respectively and decreased as cytokinin concentration increased. The antibiotic ‘Na-cefotaxime’ had a minimal impact on shoot growth when used at the lowest concentration (250 mg l⁻¹). Subculture every 2 weeks in a medium supplemented with 2.2 μM BA and 0.57 μM IAA was more efficient for shoot induction than alternation of 20 days culture in a propagation medium supplemented with 2.2 μM BA and 10 days culture in an elongation medium supplemented with 1.1 μM BA and 5.71 μM IAA. The highest number of roots/shoot (8.1) was recorded at 19.6 μM IBA.     

Propagation of rose speciesin vitro

Summary Several rose species (Rosa rugosa, R. wichuraiana, R. setigera, R. laevigata, R. banksiae, R. roxburghii, R. odorata) and interspecific hybrids were cultured to determine the appropriate concentrations of nutrients and growth regulators for shoot proliferation and root initiation. Cultured shoot tips and lateral buds from different genotypes proliferated multiple shoots on a basal medium [Murashige and Skoog (MS) salts, vitamins, glycine, sucrose, and agar] supplemented with 0 μM to 17.8 μM (4 mg·l⁻¹) 6-benzyladenine (BA) and 0 μM to 0.54 μM (0.1 mg·l⁻¹) naphthalene, acetic acid (NAA). The ability of the explants to proliferate shoots and initiate roots was affected by the genotype, the nodal position of the explant, the strength of the MS basal salts, and the growth regulators used. The buds nearest the apex exhibited the slowest rate of development. Most species had the highest shoot proliferation when cultured on basal MS medium supplemented with 8.9 μM (2 mg·l⁻¹) BA, but the degree varied by species. Root development was enhanced by lowering the concentration of MS salts. With difficult-to-root species, rooting was improved by supplementing the media with 11.4 μM (2 mg·l⁻¹) indole-3-acetic acid (IAA) or by giving them a 7-d dark treatment at 10°C.     

Direct shoot regeneration from leaf segments of mature plants of <Emphasis Type="Italic">Echinacea purpurea</Emphasis> (L.) moench

Summary This is the first communication of direct shoot regeneration from fully developed leaves of potted mature Echinacea purpurea plants. Shoot buds were induced directly on the adaxial surface of mature leaf tissues of E. purpurea 30 d after culture initiation on Woody Plant Medium (WPM) supplemented with various levels of 6-benzyladenine (BA). Maximum shoot organogenesis, with 12–20 shoots per leaf segment, was obtained with 5% coconut milk and 2.5 mg l⁻¹ (6μM) BA in 30 d. Callus was induced using 0.5 mgl⁻¹ (1μM) α-naphthaleneacetic acid and 2.5 mgl⁻¹ (6μM) BA. The regenerated shoots were rooted on WPM supplemented with 1.5 mgl⁻¹ (3μM) of indole-3-butyric acid, 3% sucrose, and 0.85% agarose. Rooted plants were successfully transferred to soil in pots and appeared morphologically normal and flowered in a growth chamber.     

Plant regeneration from excised hypocotyl explants of <Emphasis Type="Italic">Platanus acerifolia</Emphasis> willd

Summary A method of plant regeneration from hypocotyl segments of Platanus acerifolia Willd, has been developed. Hypocotyl slices were cultured on Murashige and Skoog (MS) basal medium supplemented with a range of combinations of cytokinins [6-benzyladenine (BA) or kinetin] and auxins [indole-3-butyric acid (IBA), indole-3-acetic acid, α-naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid] for adventitious shoot induetion. The highest regeneration frequency was obtained with MS medium containing 2.0 mg l⁻¹ (8.88 μM) BA and 0.5 mg l⁻¹ (2.46 μM) IBA. Adventitious buds and shoots were differentiated from hypocotyl-derived cellus or directly from the wounded sites within 4–8 wk. The regenerated shoots were elongated and proliferated efficiently on multiplication medium. Complete plantlets were transplanted to the soil and grew normally in the greenhouse after root formation on rooting medium for 4–6 wk.     

Micropropagation of <Emphasis Type="Italic">Penthorum chinense</Emphasis> through axillary bud

This study reports a protocol for successful micropropagation of Penthorum chinense using nodal explants on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine (BA) or kinetin (Kn). The presence of BA promoted a higher rate of shoot multiplication than Kn. Maximum multiple shoot formation was observed in 59.2% of nodal explants cultured on MS medium supplemented with 2.0 mg l⁻¹ BA after 6 wk. After subculture for 4 wk, the maximum number of shoots (6.4) was obtained on a medium with 2.0 mg l⁻¹ BA, but shoots were too short and not suitable for micropropagation. The taller shoots that regenerated in the presence of lower BA concentration (1.0 mg l⁻¹) were selected for root induction study. Most shoots (98.8%) rooted in the presence of 0.5 mg l⁻¹ indole-3-acetic acid after 3 wk, with each shoot forming an average of 10.0 roots. Plantlets were transferred to soil and successfully acclimatized.     

A simple method for mass propagation of Spathiphyllum cannifolium using an airlift bioreactor

Summary A method for the micropropagation of Spathiphyllum cannifolium is presented using shoot tip proliferation onto Murashige and Skoog (MS) medium supplemented with different plant growth regulator concentrations and combinations. The proliferation responses were significantly influenced by the cytokinin type and concentrations. Supplementation of the medium with benzyladenine (BA; 4.44–13.32 μM) increased the shoot proliferation rate significantly as compared to other treatments. When cytokinins were used with auxin (indole-3-butyric acid, IBA and naphthalene acetic acid. NAA), the number of shoots per explant increased in comparison with treatments with BA alone. The largest number of shoots, 9.3 per explant, was obtained with 13.32 μM BA and 4.9 μM IBA. Different MS medium strengths and sucrose concentrations were used with the aim to stimulate in vitro shoot proliferation. Full MS medium with 30 gl⁻¹ sucrose was found to be suitable for shoot tip culture of Spathiphyllum. Comparative studies between gelled medium and bioreactor culture [continuous immersion (with or without net) and temporary immersion in liquid media using ebb and flood] revealed that shoot multiplication and growth were more efficient in continuous immersion (with net) bioreactor with low cytokinin-supplemented media. Plantlets from the bioreactor were cultured hydroponically for 30 d and 100% of plants were rooted and acelimatized successfully. Rapid and efficient multiplication rate in bioreactor and successful transfer to greenhouse makes this protocol suitable for large-scale multiplication of this important foliage plant.     

Callus induction and plantlet regeneration in <Emphasis Type="Italic">Withania somnifera</Emphasis> (L.) dunal

Summary Callus induction was observed from hypocotyl, root, and cotyledonary leaf segments, grown on Murashige and Skoog (MS) medium supplemented with various concentrations and combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (KN). Maximum callusing (100%) was obtained from root and cotyledonary leaf segments grown on MS medium supplemented with a combination of 2 mg l⁻¹ (9.1 μM) 2,4-D and 0.2 mg l⁻¹ (0.9 μM) KN. The calluses, when subcultured in the same medium, showed profuse callusing. However, these calluses remained recalcitrant to regenerate regardless of the quality and combinations of plant growth regulators in the nutrient pool. When hypocotyl segments were used as explants, callus induction was noticed in 91% of cultures which showed shoot regeneration on MS medium supplemented with 2 mg l⁻¹ 2,4-D and 0.2 mg l⁻¹ KN. These shoots were transferred to fresh medium containing various concentrations and combinations of 6-benzyladenine (BA) and N⁶-(2-isopentenyl)adenosine (2-iP). Maximum shoot multiplication was observed after 60 d of the second subculture on MS medium containing 2 mg l⁻¹ (8.9 μM) BA. These shoots were rooted best (87%) on MS medium containing 2 mg l⁻¹ (9.9 μM) indole-3-butyric acid (IBA). The plantlets were transferred to the field after acclimatization and showed 60% survival.     

The development of an efficient cultivar-independent plant regeneration system from callus derived from both apical and non-apical root segments of garlic (<Emphasis Type="Italic">Allium sativum</Emphasis> L.)

Summary Callus induction and later plant regeneration were studied in four widely grown garlic (Allium sativum L.) cultivars from Europe. Root segments from in vitro plantlets were used as starting material. In addition to cultivar effects, the effects of auxin and cytokinin levels and the position of the segments on the root were studied. There were no statistically significant differences among cultivars for the number of root segments that induced callus in the two series of experiments. The average induction frequency was 34.7% in the first series of experiments. Callus induction on apical root segments was significantly higher compared to callus induction on non-apical root segments in the second series of experiments. Two months after callus induction, callus lines were transferred to a regeneration medium consisting of Murashige and Skoog basal medium supplemented with 30gl⁻¹ sucrose and 1 mgl⁻¹ (4.6μM) kinetin. Calluses derived from different experiments were quite uniform with respect to their regeneration potential. Also it was found that our regeneration system was cultivar-independent. The average shoot regeneration frequency was 17.9% in the first series of experiments. Highly significant differences were found in the frequency of shoot regeneration among different callus induction treatments. When the cytokinin 6-(γ,γ-dimethylallylamino)purine (0.1mgl⁻¹∶0.5 μM) was present during callus induction, shoot regeneration ranged from 30.10 to 47.60%. Shoot regeneration from callus induced on non-apical segments was higher, although not significant, compared to callus induction from apical root segments in the second series of experiments. All in all, an efficient callus induction and plant regeneration system was developed from both apical and non-apical segments taken along the entire length of the roots. This system has potential to be used for garlic transformation.     

Influence of some plant growth regulators on the growth and organogenesis ofCymbidium aloifolium (L.) Sw. seed-derived rhizomesin vitro

Summary An efficient procedure is outlined forin vitro regeneration of an epiphytic orchid,Cymbidium aloifolium (L.) Sw. using rhizomes developed from seeds. Murashige and Skoog's (1962) medium (MS) containing indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), or 1-naphthaleneacetic acid (NAA) stimulated growth and proliferation of rhizomes with NAA being most effective at 5.0 mg.l⁻¹ (27.0 μM). Shoot bud differentiation was induced in the apical portions of the rhizomes on MS medium containing kinetin (Kn) or N⁶-benzyladenine (BA). The highest frequency of shoot regeneration (91.5%) and the maximum number of shoot buds formed (3.5 shoots/rhizome) were recorded with BA at 1.0 mg.l⁻¹ (4.4 μM). NAA (0.1 mg.l⁻¹, 0.54 μM), whenever added to the medium in conjunction with BA (1.0 mg.l⁻¹, 4.4 μM), slightly enhanced the frequency of shoot bud regeneration (92.6%) and the number of shoot buds formed (5.2 shoots/rhizome). Moreover, an NAA-BA combination induced rooting in regenerated shoots thereby producing complete plantlets in one step. Shoots developed on cytokinin-supplemented medium were rooted on MS containing NAA at 1.0 mg.l⁻¹ (5.4 μM). Regenerated plantlets were acclimated and eventually established in a garden.     

High-frequency plant regeneration by <Emphasis Type="Italic">in vitro</Emphasis> shoot proliferation in cotyledonary node explants of grasspea (<Emphasis Type="Italic">Lathyrus sativus</Emphasis> L.)

Summary Multiple shoots were induced from cotyledonary nodes of grasspea (Lathyrus sativus L.) derived from 7-d-old in vitro seedlings on Murashige and Skoog (MS) medium containing N⁶-benzyladenine (BA), kinetin, or thidiazuron, BA being the most effective. Among the five genotypes tested, shoot proliferation frequency was the highest (93.3%) for IC-120487, giving the maximum number of shoots (11.3 shoots per explant) on MS medium augmented with 2.0 mgl⁻¹ (8.87 μM) BA. Shoot cultures were established by repeatedly subculturing the original cotyledonary nodes on fresh medium after each harvest of the newly formed shoots. Thus 30–40 shoots were obtained in 2 mo. from a single cotyledonary node. Up to 81.8% of the shoots developed roots following transfer to half-strength MS medium containing 0.5 mgl⁻¹ (2.85 μM) indole-3-acetic acid. Plantlets were successfully acclimatized and established in soil.     

High frequency plant regeneration from the cotyledonary node of common bean

An efficient regeneration system for Phaseolus vulgaris was developed from mature seeds germinated on Murashige and Skoog (MS) medium supplemented with thidiazuron or N⁶-benzylaminopurine (BA) for 6 d. Using cotyledonary nodes, multiple buds were induced on the MS medium supplemented with 5.0 mg dm⁻³ BA with the induction frequency 71.9 % after 4-week culture. The buds were then transferred onto shoot formation medium containing 1.0 mg dm⁻³ BA, 0.1 mg dm⁻³ gibberellic acid and 2.0 mg dm⁻³ silver nitrate. The addition of AgNO₃ enhanced the frequency of the shoot formation from 61.3 to 87.6 %. Root induction medium was half-strength MS medium with 0.75 mg dm⁻³ indolebutyric acid and 0.02 mg dm⁻³ BA. The average root frequency was 84.3 %. The regenerated plantlets with healthy roots grew successfully when transferred to soil. Using this system we obtained over 10 regenerated plantlets from one explant.     

High-frequency callus induction and plant regeneration in Tripsacum dactyloides (L.)

Summary A protocol for high-frequency callus, somatic embryogenesis, and plant regeneration for Tripsacum is described. Plants were regenerated from complete shoot meristems (3–4 mm) via organogenesis and embryogenesis. In organogenesis, the shoot meristems were cultured directly on a high cytokinin medium comprising 5–10 mgl⁻¹ (22.2–44.4 μM) 6-benzyladenine (BA). The number of multiple shoots varied from six to eight from each meristem. The time required for production of plants from organogenesis was rapid (4–6 wk). In contrast, callus was induced on an auxin medium and continuously cultured on an auxin medium for production of somatic embryos. Prolific callus with numerous somatic embryos developed within 3–4 wk when cultured on an auxin medium containing 5 mgl⁻¹ (22.6μM), 2,4-dichlorophenoxyacetic acid (2,4-D). The number of shoots induced varied from two to five per callus. Regardless of the cultivars used, the frequency of callus induction and plant regeneration was between 48% and 94%. The seed germination procedures also were modified and resulted in a maximum of 60–80% seed germination. Finally, the rate of T-DNA transfer to complete shoot meristems of Tripsacum was high on the auxin medium and was independent of whether super-virulent strains of Agrobacterium were used or not.     

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.     

Micropropagation of <Emphasis Type="Italic">Leptadenia reticulata</Emphasis>—A medicinal plant

Summary Leptadenia reticulata (Retz.) Wight. & Arn., an important herbal medicinal plant, belongs to the family Asclepiadaceae. This plant has been known for its medicinal uses since 4500 BC. Presently this is an endangered species. There is a need for applying non-conventional methods of propagation for conservation and sustainable utilization of biodiversity of Leptadenia reticulata. We developed a micropropagation method for mass multiplication of L. reticulata. Explants harvested from greenhouse-maintained and field-grown plants were used to establish cultures of L. reticulata. The nodal shoot segments were surface-sterilized and cultured on Murashige and Skoog (MS) medium along with additives (25 mg l⁻¹ each of adenine sulfate, arginine, and citric acid, and 50 mg l⁻¹ ascorbic acid) containing 0.6 μM indole-3-acetic acid (IAA) and 9 μM N⁶-benzyladenine (BA). Three to four shoots differentiated from each node within 25–30 d at 26±2°C and 36 μmol m⁻² s⁻¹ spectral flux photon (SFP) for 12 hd⁻¹. Shoots were further multiplied by (1) repeated transfer of mother explant on fresh medium containing 0.6 μM IAA and 2.2 μM BA, and (2) subculture of in vitro-differentiated shoots on MS medium with 6.6 μM BA and 0.6 μM IAA. After three or four subcultures, the basal clump with shoot bases was divided into three or four subclumps and multiplied on the fresh medium. From each clump 15–20 shoots regenerated within 25 d. Ninety percent of the in vitro-produced shoots rooted ex vitro if these were pulse-treated with 123 μM each of indole-3-butyric acid and β-naphthoxyacefic acid. The plantlets were transferred to bottles containing sterile ‘soilrite’ (soilless compost and soil conditioner) moistened with half-strength MS macrosalts. Ninety-five percent of the plantlets were hardened in the bottles within 15 d. The hardened plants were then transferred to black polybags in the nursery. Field transferred plants are growing normally and have flowered. The protocol developed is reproducible. From a single nodal segment about 1700 hardened plants could be regenerated within 174 d.     

Tissue culture and plant regeneration of blue grama grass, Bouteloua gracilis (H.B.K.) Lag. Ex Steud

Summary As a first step towards applying biotechnology to blue grama, Bouteloua gracilis (H. B. K.) Lag. ex Steud., we have developed a regenerable tissue culture system for this grass. Shoot apices were isolated from 3-d-old seedlings and cultured in 15 different growth regulator formulations combining 2,4-dichlorophenoxyacetic acid (2,4-D), Picloram (4-amino-3, 5,6-trichloropicolinic acid), N⁶-benzyladenine (BA) or adenine (6-aminopurine). The highest induction of organogenic callus was obtained with formulations containing 1 mg l⁻¹ (4.52 μM) 2,4-D plus 0.5 mg l⁻¹ (2.22 μM) BA. and 2 mg l⁻¹ (8.88 μM) BA plus 1 mg l⁻¹ (4.14 μM) Picloram with or without 40 mg l⁻¹ (296.08 μM) adenine. Lower frequencies of induction were obtained for embryogenic as compared to organogenic callus. The most efficient treatments for induction of embryogenic callus contained 2 mg l⁻¹ (9.05 μM) 2,4-D combined with 0.25 (1.11 μM) or 0.50 mg l⁻¹ (2.22 μM) BA, or 1 mg l⁻¹ (4.52 μM) 2,4-D with 0.50 mg l⁻¹ (2.22 μM) BA. Regeneration was achieved in hormonefree Murashige anmd Skoog (MS) medium, half-strength MS medium or MS medium plus 1 mg l⁻¹ (1.44 μM) gibberellic acid. The number of plantlets regenerated per 500 mg callus fresh weight on MS medium ranged from 9 for 2 mg l⁻¹ (9.05 μM) 2,4-D to 62.2 for induction medium containing 2 mg l⁻¹ (8,28 μM) Picloram, 1 mg l⁻¹ (4.44 μM) BA and 40 mg l⁻¹ (296.08 μM) adenine. Regnerated plants grown in soil under greenhouse conditions reached maturity and produced seeds.     

Micropropagation ofCereus peruvianus mill. (cactaceae) by areole activation

Summary Currently,Cereus peruvianus plants can be rapidly clonedin vitro via adventitious organogenesis using callus cultures; however, somaclonal variation is a problem. A method is described herein using lateral bud explants to produce multiple shoots for clonal propagation. Apical and lateral explants were cultured on MS (Murashige and Skoog, 1962) media with factorial combinations of the auxins indole-3-acetic acid (IAA), 1-naphthaleneacetic acid (NAA), and cytokinins 6-ben-zyladenine (BA) and N-(2-furanyl-methyl)-1-purine-6 amine (kinetin) at the concentrations 0.0, 0.01, 0.1, 1.0 mg“l⁻¹. Positive results were obtained from the lateral explants in all conditions tested, but apical explants did not respond toin vitro multiplication ofC. peruvianus cactus at all growth regulator combinations tested. Formation of axillary shoots inC. peruvianus seems most frequent in medium containing BA at 1.0 mg·l⁻¹ (4.44 μM) and IAA or NAA at 1.0 mg·l⁻¹ (5.71 μM or 5.37 μM respectively), but the frequency of shoot formation in the BA or kinetin and NAA or IAA combinations indicated that any of the combinations tested can be used for multiplication ofC. peruvianus plants regenerated from callus tissue culture. Root formation occurred in all (100%) of the cactus shoots after 9 wk in the same culture medium. All the cacti that developed at the different auxin and cytokin combinations continued growth after transfer to a potting mix of red earth (Paleudult) and ground river sand (1∶1).     

Micropropagation studies inCeropegia SPP

Summary The purpose of this study was to developin vitro techniques for conserving wild and endemic species ofCeropegia by mass multiplication for subsequent reintroduction in their natural habitat. Micropropagation involving a combination of axillary bud culture, shoot multiplication, somatic embryogenesis andin vitro tuber formation forCeropegia jainii, a rare plant of the Indian sub continent,C. bulbosa var.bulbosa andC. bulbosa var.lushii, common species, was developed. Nodal explants from all species were cultured on 0.5 MS medium with 8.8 μM (2 mg·l⁻¹) N⁶-benzyl aminopurine (BA) to regenerate the axillary buds. These produced multiple shoots when transferred to multiplication medium consisting of 0.5 MS medium with 2.2 μM (0.5 mg·l⁻¹) BA, or microtubers when transferred to 0.5 MS medium with 22.2 μM (5 mg·l⁻¹) BA and 23.2 μM (5 mg·l⁻¹) kinetin.In vitro flowering occurred inC. jainii and not in the other two varieties when the plants were cultured on multiplication media with spermine at 0.25 μM (50 μg·l⁻¹) as an additive. Shoot pieces produced callus on MS medium with 9.05 μM (2 mg·l⁻¹) 2,4-dichlorophenoxy acetic acid. Regeneration of the calli by somatic embryogenesis was achieved when they were transferred to 0.5 MS medium with 2.2 μM (0.5 mg·l⁻¹) BA. Rooting of the shoots was possible both byin vitro andex vitro means.     

Tissue culture of goldenseal (Hydrastis canadensis L.)

Summary Goldenseal (Hydrastis canadensis L.), a popular native American medicinal plant, is currently listed as endangered or threatened in over one-third of the states in which it is listed. The objective of this study was to develop an in vitro culture protocol for Goldenseal. Excise embryos were grown on Gamborg's B-5 medium with 0,1 or 10 μM gibberellic acid (GA₃), and supplemented with 30 gl⁻¹ sucrose and 8 gl⁻¹ agar. Germinated embryos provided explants (leaf and root tissue) that were subsequently cultured on various media with combinations of naphthleneacetic acid (NAA) and benzyladenine (BA). All NAA/BA combinations produced multiple shoots, roots, and callus. Leaf explants cultured on medium with 1∶10 μM NAA:BA and root explants on medium with 1∶1 μM NAA:BA could be successfully used for mieropropagation.     

Cytokinins,auxins and activated charcoal affect organogenesis and anatomical characteristics of shoot-tip cultures of lisianthus [<Emphasis Type="Italic">Eustoma grandiflorum</Emphasis> (RAF.) Shinn]

Summary Variants from seed-propagated Lisianthus [Eustoma grandiflorum (Raf.) Shinn] were shoot-tip cultured to observe the effects of cytokinins, auxins and activated charcoal on organogenesis and anatomical characteristics. N⁶-Benzyladenine (BA) and kinetin at high concentrations (13.32–22.2 and 13.94–23.23 μM) resulted in good shoot formation but high percentages of hyperhydric shoots. Increased indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) concentrations favored root formation, while increased naphthaleneacetic acid concentration adversely affected root formation. Both shoot and root development were suppressed by activated charcoal. The highest percentage of regeneration and the largest number of glaucous shoots with an average of 15 shoots per explant after 4 wk of culture were obtained when the shoot tips were cultured on MS (Murashige and Skoog, 1962) medium supplemented with 4.44 μM BA and 1.47–4.92 μMIAA and IBA. In vitro-grown leaves had a higher number of stomata than field-grown leaves but the length and diameter of stomata showed no significant difference between the two types. Field-grown leaves had well-developed epicuticular wax layers. which were not observed on hyperhydric leaves. Hyperthydric plantlets could not survive when transplanted to soil, whereas glaucous plantlets survived in more than 80% of cases. Variation in soil type resulted in a slight difference in plantlet survival. Based on the results of our experiment, this protocol should be useful for the rapid micropropagation of lisianthus.