Plant regeneration from somatic tissue of Rhododendron laetum x aurigeranum

The influence of the source of plant material (greenhouse-grown plants or in vitro shoot cultures), the type of tissue explant (shoot-tip, single-node stem segment, whole leaf, leaf strip or half-leaf section) and growth regulator concentration on shoot regeneration from somatic tissue of Rhododendron laetum × aurigeranum was evaluated. No regeneration response was obtained on explants from greenhouse-grown plants. Adventitious shoots were obtained from callus produced at the basal end of shoot-tip and single-node stem segment explants derived from in vitro-grown shoots cultured on Anderson's medium supplemented with 22.8 M IAA and 73.8 M 2iP. The greatest percentage of adventitious shoot regeneration (77%) was induced on leaf sections cultured in the presence of 22.8 M IAA and 147.6 M 2iP. Plant regeneration was accomplished with minimal callus formation. This technique represents a further step toward gene manipulation of Rhododendron.Abbreviations IAA 1-H-Indole-3-acetic acid - 2iP N-(3-methyl-2-Butenyl)-1H-purin-6 amine     

Rapid adventitious shoot regeneration from leaf explants of European birch

The goal of this research was to develop a rapid and efficient system for regenerating shoots from leaf explants of European birch, Betula pendula Roth. Single-node stem explants were established in culture, and microshoots were subcultured every 4 weeks through 12 subcultures. Leaves from glasshouse plants or subcultured shoots were excised from stems, cut into approximately 35-mm² pieces, and placed on Woody Plant Medium (WPM) containing different combinations of naphthaleneacetic acid (NAA) (0, 3, 6 or 9 M) and benzyladenine (BA) (0, 7.5, 15 or 22.5 M) in a 4×4 factorial design. The percentage of leaf pieces forming shoots and the number of shoots regenerated per explant were recorded after 4 weeks. Only media containing BA without NAA stimulated shoot formation on leaf explants. Fifteen micromolar BA induced the most shoots to form on leaf explants compared to 30, 45 or 60 M of this cytokinin. In addition, shoot regeneration was enhanced up to four-fold between the first and eleventh subculture. Over 90% of the leaf explants regenerated shoots with an average of 18 buds formed per explant for the eleventh subculture. Almost twice as many explants formed shoots if their adaxial side was in contact with the medium rather than oriented away from it. The ability to regenerate shoots from leaves varied among plants, regardless of stock plant age. This reliable shoot regeneration system can be used for rapid shoot proliferation and potentially for genetic engineering of European birch.     

Cells within the nodal region of carnation shoots exhibit a high potential for adventitious shoot formation

Adventitious shoot formation was studied with leaf, stem and axillary bud explants of carnation (Dianthus caryophyllus L.). The shoot regeneration procedures were applicable for a wide range of cultivars and shoot regeneration percentages were high for all explant types. Using axillary bud explants, shoot regeneration efficiency was independent of the size of the bud and of its original position in the plant. In contrast, shoot regeneration from stem and leaf explants was strongly dependent on their original position on the plant. The most distal explants (just below the apex) showed the highest level of shoot regeneration. The adventitious shoot primordia developed at the periphery of the stem segment and at the base of leaf explants. In axillary bud, stem and leaf explants, shoot regeneration originated from node cells, located at the transition area between leaf and stem tissue. Moreover, a gradient in shoot regeneration response was observed, increasing towards the apical meristem.Abbreviations BA benzyladenine - NAA naphthaleneacetic acid     

An efficient adventitious shoot regeneration system for Zhanhua winter jujube (Zizyphus jujuba Mill.) using leaf explants

The direct induction of adventitious shoots from leaf explants obtained from adult plants of Zhanhua winter jujube, an elite variety of Zizyphus jujuba Mill., is reported. The percentage of leaf explants producing shoots and the average number of shoots per explant were significantly improved when 10-day-old leaves were explanted onto Woody Plant Medium and maintained initially in the dark. The plant growth regulator thidiazuron (TDZ) was effective in stimulating shoot regeneration from leaf explants of Zhanhua winter jujube. The highest efficiency of shoot formation was observed with a 20-day culture in the dark on WPM containing 4.54 M TDZ and 2.85 M indoleacetic acid (IAA). The regenerated shoots were transferred to MS medium containing 0.89 M benzyladenine and 5.77 M gibberellic acid for growth. When the shoots were about 2 cm in height, they were transferred to Nitsch medium supplemented with 1.14 M IAA and 2.46 M indolebutyric acid to induce rooting. This system of adventitious shoot production from leaf explants of adult plants could be useful for the genetic engineering and polyploidization of winter jujube.     

Cytokinins,donor plants and time in culture affect shoot regenerative capacity of American elm leaves

Cytokinins, donor plants and their time in vitro as well as basal media were investigated for their influence on shoot regenerative capacity of American elm (Ulmus americana L.) leaves. Leaves excised from six 2-year-old seedlings formed adventitious shoots when placed on Driver and Kuniyuki Walnut (DKW) medium supplemented with 7.5, 15 or 22.5 M of benzyladenine (BA) or thidiazuron (TDZ). Thidiazuron induced significantly higher regeneration percentages on elm leaves than BA, regardless of concentration used. Donor plant also affected the efficiency of shoot regeneration, with certain seedlings having 1.5 to 7 times more explants forming shoots as compared to other seedlings tested. By subculture 15, the average number of shoots per regenerating explant increased at least 3-fold for leaves on media with BA or TDZ for the one donor plant that survived continued subculturing. Leaf explants from donor plants with the highest regenerative capacity had a higher percentage of shoot formation on DKW than MS medium. Explants from productive donor plants should be placed on DKW medium supplemented with TDZ to improve shoot regeneration efficiency from American elm leaves.     

The effect of leaf source and developmental stage on shoot organogenic potential of sweetgum (Liquidambar styraciflua L.) leaf explants

Leaf explants harvested from shoot proliferating cultures and intact plants of Liquidambar styraciflua Variegata were placed on solidified Woody Plant medium supplemented with 0.1 mgl^-1 (0.5 M) naphthaleneacetic acid and 2.5 mgl^-1 (11.1 M) benzyladenine to initiate shoot meristems directly. Leaves from intact plants produced over 4 times more adventitious shoots than leaves from in vitro shoots and had a greater tendency to form shoots on the lamina. The relative developmental age of leaf tissue used dramatically influenced the shoot organogenic response observed for leaf explants from intact plants of L. styraciflua Variegata and Moraine.-Leaves that were either 20% or 50% of full size and still actively expanding were superior to other developmental stages for shoot organogenesis. As developmental leaf age increased throughout the period of leaf expansion, the number of shoots forming on the petiole stub remained constant, whereas shoot formation on the lamina increased 8 fold. Shoots derived from Variegata leaves rooted well and grew normally as plants. Differences in rooting ability and plant size could be detected between groups that had been separated according to explant source (in vitro vs. intact plant) and the location of shoot formation (petiole vs. lamina).     

In vitro multiplication of a sterile interspecific hybrid,Brassica fruticulosa x B. campestris

In vitro methods for plant multiplication of a sterile interspecific hybrid between Brassica fruticulosa and B. campestris through either micropropagation or callus regeneration is described. Shoot-tip, single-node and leaf explants, obtained from in vitro-grown hybrids, regenerated on media containing NAA and BA. In vitro application of colchicine induced chromosome doubling in in vitro-regenerated shoots resulting in the production of fertile amphidiploids. Comparative studies on regeneration potential of the hybrid and its parents were also carried out using callus from leaf explants. The explants of B. fruticulosa and the hybrid were capable of shoot and root formation while those of B. campestris failed to form shoots but produced profuse roots. The results demonstrate the efficacy of an in vitro method in producing a large number of hybrid plants and fertile amphidiploids from incompatible crosses that yield very few hybrid seeds/seedlings.Abbreviations BA benzyladenine - CMS cytoplasmic male sterile - AA diploid genome of B. campestris - FF diploid genome of B. fruticulosa - NAA -naphthaleneacetic acid     

Direct plant regeneration from leaf explants of Plumbago species

Direct plant regeneration was achieved from leaf explants of Plumbago rosea and Plumbago zeylanica on Murashige and Skoog (MS) medium supplemented with 6.7 M 6-benzylaminopurine (BA), 1.4 M indole-3-acetic acid (IAA), 370 M adenine sulfate (Ads) and 3% (w/v) sucrose. The shoot initials developed within 2–3 weeks on the leaf margin as well as from the cut surface of the leaf. High frequency shoot-bud regeneration was achieved on similar medium in subsequent subcultures. The semi-mature leaves produced more shoot-buds as compared to the younger leaves. Mature leaves did not show any response for shoot bud initiation. More than 85% of the semi-mature explants produced shoot-buds per leaf explant within 4 weeks of culture. Shoots rooted on half-strength basal MS medium supplemented with 1.2 M indole-3-butyric acid (IBA) and 2% (w/v) sucrose; approximately 90% of the in vitro raised plantlets survived in the greenhouse. The regenerated plantlets looked morphologically similar to the mother plants. This protocol might be useful for genetic improvement programs.     

Transgenic carnations obtained byAgrobacterium tumefciens-mediated transformation of leaf explants

For the development of anAgrobacterium-mediated transformation procedure of carnation (Dianthus caryophyllus L.), an intron-containing -glucuronidase (gus) gene was used to monitor the frequency of transformation events soon after infection of leaf explants. The efficiency of gene transfer was dependent on the carnation genotype, explant age and cocultivation time. Leaf explants from the youngest leaves showed the highest number of GUS-positive spots. After selection on a kanamycin-containing medium, transgenic shoots were generated among a relatively high number of untransformed shoots. The selection procedure was modified in such a way that the contact between explant and medium was more intense. This improved the selection and decreased the number of escapes. Kanamycin-resistant and GUS-positive plants were obtained from five cultivars after infection of leaf explants with the supervirulentAgrobacterium strain AGLO. A higher transformation frequency was observed with the binary vector pCGN7001 than with the p35SGUSint vector. Integration of the genes into the carnation genome was demonstrated by Southern blot hybridization. The number of incorporated T-DNA insertions varied between independent transformants from one to eight. Transformants were morphologically identical to untransformed plants. Segregation of the genes occurred in a Mendelian way.     

Effect of genotype,explant, subculture interval and environmental conditions on regeneration of shoots from in vitro monoploids of a diploid potato species,Solanum phureja Juz. & Buk.

In vitro anther-derived monoploids (2n=x=12) of Solanum phureja were compared for shoot regeneration from leaf and stem explants under various environmental conditions. Monoploids from the same or different diploid clones varied for frequency and earliness of shoot regeneration and number of shoots formed per explant. Leaf explants regenerated at higher frequencies than stem explants. Explants from stock plantlets subcultured at a 2- or 4-week interval regenerated earlier and at a higher frequency than those from plantlets subcultured at longer intervals. Regeneration frequency and number of shoots per explant were greater when explants were incubated at 20°C compared to 25°C. Explants from stock plantlets maintained under a 16 h as opposed to an 11 h photoperiod exhibited increased shoot regeneration; however, neither photoperiod nor the maintenance temperature of the stock plantlets influenced regeneration frequency. Genotypic differences were observed for the frequency of chromosome doubling among regenerated shoots whereas temperature treatments had no influence on chromosome doubling.Abbreviations BA benzyladenine - GA₃ gibberellic acid - IAA indole-3-acetic acid - NAA -naphthale-neacetic acid     

Efficient plant regeneration from leaves of rapeseed (Brassica napus L.): the influence of AgNO3 and genotype

Factors influencing reliable shoot regeneration from leaf explants of rapeseed (Brassica napus L.) were examined. Addition of AgNO₃ to callus induction medium was significantly effective for shoot regeneration in all three genotypes initially tested. When 48 genotypes subsequently were surveyed, a large variation of shoot regenerability was observed, ranging from 100 to 0% in frequency of bud formation and from 7.5 to 0 in the number of buds per explant. A significant correlation (r=0.84) was observed between the frequency of bud formation and the number of buds per explant. The shoot regenerability from leaf explants was not related to that from cotyledonary explants (r=0.28). Histological observations showed that an organized structure developed from calluses produced at vascular bundle tissues after 7 days of culture on callus induction medium, and they developed shoot apical meristems one week after transfer onto shoot induction medium. Regenerated plantlets were obtained 2 months after the initiation of culture and they normally flowered and set seeds. No alterations of morphology or DNA contents were observed in regenerated plants and their S1 progenies.     

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

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

In vitro regeneration of Echinacea purpurea from leaf explants

Efficient plant regeneration was achieved via organogenesis from callus cultures derived from leaf tissue of Echinacea purpurea. Proliferating shoot cultures were obtained by placing leaf explants on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (BAP) and naphthaleneacetic acid (NAA) combinations. MS medium supplemented with BAP (4.44 M) and NAA (0.054 M) was the most effective, providing high shoot regeneration frequencies (100%) associated with a high number of shoots per explant (7.7 shoots/explant). Plantlets were rooted on MS medium alone or in combination with different concentrations of indole-3-butyric acid (IBA), and high rooting and survival was achieved using MS media without plant growth regulators (PGR). All plantlets survived acclimatization producing healthy plants in the greenhouse. This study demonstrated that adventitious shoot regeneration of E. purpurea from leaf explants can be a useful method for the multiplication of this important medicinal plant.     

Shoot production per responsive leaf explant increases exponentially with explant organogenic potential in Nicotiana species

As part of the effort to develop optimal plant varieties for the production and molecular farming of plant-made pharmaceuticals, this study evaluated shoot organogenic potential of a total of 115 Nicotiana accessions, representing 53 species. To induce shoots, leaves from seedling grown in vitro were cut into pieces, cultured on shoot-induction medium under low light for 3 weeks, and then subcultured onto the same medium for another 4 weeks under normal light. Statistical analysis detected significant differences among the 115 accessions for the percentage of leaf explants producing shoots and the number of shoots produced per responsive leaf explant. Importantly, regression analysis also found an exponential relationship between the number of shoots produced per responsive leaf explant and the percentage of leaf explants producing shoots. The number of shoots produced per responsive leaf explant increased rather slowly, ranging from zero to around five, as the percentage of leaf explants producing shoots increased from 0 to 80%, but the increase became dramatic as the percentage increased from 80% to 100%, reaching as high as 35 shoots per responsive leaf explant. This exponential relationship is the first of its kind to be established in plant regeneration studies using either organogenesis or somatic embryogenesis systems. A possible mechanism that governs the establishment of the exponential relationship is discussed.Abbreviations 2ip 6-(,-Dimethylallylamino)-purine - BA Benzylaminopurine - IAA Indole-3-acetic acid - LS Linsmaier and Skoog - MS Murashige and Skoog - PI Plant introduction number - PMP Plant-made pharmaceuticals - SIM Shoot induction medium - USDA US Department of Agriculture     

Pretreatment in thidiazuron improves the in vitro shoot induction from leaves in Curculigo orchioides Gaertn., an endangered medicinal plant

Leaf regeneration via direct induction of adventitious shoots obtained from an endangered medicinal plant, Curculigo orchioides Gaertn. by pretreating with thidiazuron. C. orchioides is an endangered medicinal herb belonging to the family Hypoxidaceae. Direct inoculation of leaf pieces on MS medium supplemented with various concentrations of BAP (2–8 μM) or TDZ (2–8 μM) alone or in combination with NAA (0.5 and 1.0 μM) produced low shoot induction both in terms of % response and number of shoots per explant. Hence, leaf explants were pretreated with 15, 25 or 50 μM thidiazuron (TDZ), for 6, 24 or 48 h with the aim of improving shoot regeneration from cultured explants. After pretreatment, explants were transferred to an agar solidified MS medium that was supplemented with BAP (4 μM), TDZ (6 μM), BAP (4 μM) + NAA (1.0 μM), TDZ (6 μM) + NAA (0.5 μM). Control explants were incubated directly on the medium without any pretreatment. The pretreatment of explants with 15 μM TDZ for 24 h significantly promoted the formation of adventitious shoots and the maximum response was observed on MS medium supplemented with 6 μM TDZ. In this medium, 96 % cultures responded with an average number of 16.2 adventitious shoots per explant. The percentage of leaf explants producing shoots and the average number of shoots per explant were significantly improved when TDZ pretreated leaves were cultured onto MS medium supplemented with BAP or TDZ alone or in combination with NAA. The rooted plantlets were successfully transplanted to soil with 90% success. The present investigation indicated the stimulatory role of TDZ pretreatment in regulating shoot regeneration from leaf explants of C. orchioides.     

Plant regeneration from stem and petal of carnation (Dianthus caryophyllus L.)

Plants were regenerated via adventitious shoot initiation from petal explants of carnation (Dianthus caryophyllus L.) cultivars Crowley Sim, Ember Rose, Orchid Beauty, Red Sim, White Sim and from stem segments of Crowley Sim, Red Sim, White Sim. Differences in cultivar response were observed, with White Sim being the most responsive for both explant types. Plants were also regenerated from receptacles of this cultivar. The effect of different cytokinins on regeneration from petal and stem explants of cultivar White Sim was compared. Thidiazuron was more effective than 6-benzylaminopurine or kinetin. In stem explants, morphogenic capacity was determined by the developmental stage of the explant. Highest percentage of shoot formation was observed in the youngest stem segments, on all the cytokinins tested. Stem-derived plants grew faster than petal or receptacle-derived plants and produced normal, flowering plants eight to ten months after culture.     

Direct shoot regeneration from excised leaf explants of in vitro grown seedlings of Alstroemeria L.

A two-step protocol for the induction of shoots from Alstroemeria leaf explants has been developed. Leaf explants with stem node tissue attached were incubated on shoot induction medium for 10 days, and then transferred to regeneration medium. Shoots from the area adjacent to the region between the leaf base and node tissue regenerated within 3 weeks after transfer to the regeneration medium, without a callus phase. The best induction was obtained with Murashige and Skoog medium containing 10 μm thidiazuron and 0.5 μm indole butyric acid. The regeneration medium contained 2.2 μm 6-benzylaminopurine. After several subcultures of the leaf explants with induced shoots, normal plantlets with rhizome were formed. In Alstroemeria, the percentage of responding leaf explants is more important than the number of shoots regenerated per leaf explant, because rhizome formation is the most important factor for micropropagation. The effect of other compounds in the induction medium, including glucose, sucrose, silver nitrate, and ancymidol, on regeneration was also investigated. Received: 14 June 1996 / Revision received: 27 September 1996 / Accepted: 20 October 1996     

Shoot regeneration from tissue-cultured leaves of the American cranberry (Vaccinium macrocarpon)

A method for shoot regeneration from leaf explants in two cultivars of cranberry (Vaccinium macrocarpon Ait.) is described. Modified Anderson's medium supplemented with combinations of thidiazuron (TDZ) with or without 1 M NAA (-naphthaleneacetic acid) was used to optimize shoot regeneration. The effect of light or dark incubation was also determined. Maximum regeneration was obtained in the light in the presence of 10 M TDZ and 1 M NAA. While this medium was suitable for leaf explants obtained from shoot cultures, regeneration did not occur from leaves collected from greenhouse-grown plants. Elongation of the regenerated shoot tips did not occur until explants were transferred to growth regulator-free medium at which time only a minority of shoots elongated. Elongated shoots could be dissected away from leaf tissue, rooted easily, and acclimitized to ambient conditions.Abbreviations NAA -naphthaleneacetic acid - TDZ 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea     

Plant regeneration via organogenesis in marigold

Regeneration of whole plants of marigold (Tagetes erecta L.) was achieved by organogenesis using leaf explants. Leaf segments about 0.25 cm² were taken from 3-week-old in vitro plantlets and cultured on MS basal medium containing BA with different auxins (NAA, 2,4-D and IAA). The exposure time of the explants on the regeneration medium was tested. The highest values for regeneration were obtained with BA (13.3 M) and IAA (17.1 M). Thirteen days was the best time of exposure of the explant to the regeneration medium for shoot induction.     

Morpho-histological characterization and direct shoot organogenesis in two types of explants from <Emphasis Type="Italic">Bacopa monnieri</Emphasis> on unsupplemented basal medium

In the present study, high frequency regeneration has been obtained via de novo direct shoot organogenesis from leaf and internode explants in Murashige and Skoog (MS) basal medium without any phytohormone supplementation in Bacopa monnieri, an indigenous traditionally used medicinal herb. Leaves and internodes from different positions were excised from 4-weeks-old in vitro propagated B. monnieri plants and cultured on MS basal medium supplemented with 3% (w/v) sucrose and 0.75% (w/v) agar for 4 weeks. The induction of de novo shoot buds was observed at petiolar cut edges of leaf and both proximal and distal cut ends of internode explants within 10–15 days of culture. The first histological changes could be observed after 4–5 days, with meristematic activity of vascular bundles. Proliferation of epidermal cells gave rise to dome-shaped protuberances followed by shoot apical meristems formation and their vascular connections with explant tissues within 2 weeks of culture. However, a basipetal gradient of shoot regeneration from both types of explants collected along the branch axis was noticed after 4 weeks of culture. Leaf and internode explants near the basal region exhibited significantly higher number of shoot buds and micro shoots (8.8/leaf explant and 15/internode explant). Microshoots (7–12 micro shoots/leaf or internode explants) elongated (shoot length 8–9 cm) within 8 weeks on phytohormone free MS medium. Excised micro shoots rooted (100%) in hormone free MS medium within two weeks of culture. Rooted plants were then acclimatized and transferred to field with 95% survival. This protocol may be used for micropropagation, genetic transformation as well as a model system for evaluation of changes associated with acquisition of competence of differentiated cells in phytohormone free medium.