An efficient in vitro method for mass propagation of a woody ornamentalIxora coccinea L.

Various factors that affect culture establishment, shoot growth, proliferation and rooting ofIxora coccinea L., a woody shrub, were studied. Stem cuttings (decapitated shoot, three nodes) were the most suitable explants for multiple-shoot proliferation, and when cultured on a woody plant medium (WPM) containing 2.5 M BA produced axillary shoots which branched repeatedly, yielding an average of 27 shoots per explant after 6 weeks in culture. Kinetin, 2-iP, zeatin and thidiazuron all induced multiple-shoot formation, but were less effective than BA. While the presence of IAA in the multiplication medium was detrimental to shoot proliferation, shoot growth was not affected by IAA. The production of large amounts of basal callus and vitrification of shoots were the major problems to be avoided in proliferating shoot cultures. Addition of TIBA to the multiplication medium markedly reduced basal callusing, while sealing the culture vessels with a fluorocarbon polymer (tetrafluoroethyleneperfluoroalkyl vinyl ether) film (Neoflon PFA film) almost completely eliminated vitrification. A reduction in the number of vitrified shoots was also achieved with AVG treatment. Following this protocol of using BA-supplemented WPM and Neoflon film, it would be possible to produce more than 100,000 plants from a single stem cutting in 1 year.Abbreviations AVG Aminoethoxyvinylglycine - BA N⁶-benzyladenine - BM basal medium - IAA indole-3-acetic acid - IBA indole-3-butyric acid - 2-tiP N⁶-(2-isopentenyl)adenine - KIN kinetin - MS Murashige and Skoog medium - NAA -naphthaleneacetic acid - SRM shoot regeneration medium - TDZ thidiazuron - TIBA 2,3,5-triiodobenzoic acid - WPM woody plant medium - ZEA 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.     

Ectopic expression of <Emphasis Type="Italic">Dahlia merckii</Emphasis> defensin <Emphasis Type="Italic">DmAMP</Emphasis>1 improves papaya resistance to <Emphasis Type="Italic">Phytophthora palmivora</Emphasis> by reducing pathogen vigor

Phytophthora spp., some of the more important casual agents of plant diseases, are responsible for heavy economic losses worldwide. Plant defensins have been introduced as transgenes into a range of species to increase host resistance to pathogens to which they were originally susceptible. However, the effectiveness and mechanism of interaction of the defensins with Phytophthora spp. have not been clearly characterized in planta. In this study, we expressed the Dahlia merckii defensin, DmAMP1, in papaya (Carica papaya L.), a plant highly susceptible to a root, stem, and fruit rot disease caused by Phytophthora palmivora. Extracts of total leaf proteins from transformed plants inhibited growth of Phytophthora in vitro and discs cut from the leaves of transformed plants inhibited growth of Phytophthora in a bioassay. Results from our greenhouse inoculation experiments demonstrate that expressing the DmAMP1 gene in papaya plants increased resistance against P. palmivora and that this increased resistance was associated with reduced hyphae growth of P. palmivora at the infection sites. The inhibitory effects of DmAMP1 expression in papaya suggest this approach has good potential to impart transgenic resistance against Phytophthora in papaya.     

Micropropagation ofPyrus andCydonia and their responses to Fe-limiting conditions

Appropriate micropropagation regimes were determined for fourPyrus species (P. amygdaliformis Vill.,P. betulaefolia Bunge,P. calleryana Dene., andP. communis L.) andCydonia oblonga L. Shoot multiplication was optimal at 10 or 20M N⁶-benzyladenine and high light intensity (135E m^–2 s^-1). Root formation of thePyrus species was stimulated by exposure of shoots to high levels (10 or 32M) of-indolebutyric acid (IBA) for 7 days or a dip in 10 mM IBA for 15 s, followed by a passage on auxin-free medium.-Naphthaleneacetic acid was more effective than IBA in stimulating rooting ofC. oblonga. The effects of Fe-limiting conditions in vitro were determined by comparing the responses of shoots and rooted plantlets to medium containing FeEDTA or FeSO₄, with or without bicarbonate. Symptoms of Fe deficiency were genotype-dependent and most severe in the presence of FeSO₄ and bicarbonate. Chlorosis was pronounced inCydonia, absent fromP. amygdaliformis andP. communis, and intermediate inP. betulaefolia andP. calleryana, indicating a good correlation between in vitro and field responses. Similar responses were obtained with rooted and unrooted shoots. These findings suggest that in vitro cultures may be used for studies of Fe-chlorosis as well as screening for tolerant rootstocks.     

Micropropagation of Calluna vulgaris cv. ‘H.E. Beale’

Axillary shoot producing cultures were obtained from microcuttings and shoot tips of Calluna vulgaris cv. H.E. Beale. For cultures derived from microcuttings the highest multiplication rate of 38 shoots (5 mm or longer) was obtained on a reduced salt medium with the addition of 0.5 mgl^-1 2-isopentenyladenine (2iP) during an 8 week subculture. For shoot tip derived cultures 0.2 mgl^-1 6-benzyladenine (BA) was the best cytokinin and led to a multiplication rate of 26 for a 6 week subculture. The addition of 1 g/l casein hydrolysate to a multiplication medium enhanced shoot proliferation in presence of 0.5 mgl^-1 BA.Despite various auxin treatments shoots formed no roots in vitro but rooted readily if transferred to a peat substrate ex vitro. A high rooting percentage (80%) was also obtained with shoots taken from the end of a multiplication phase and rooted directly. An additional subculture on low auxin containing media before transfer to peat substrate is recommended because the shoot condition can be improved in this way. A high number of rooted plantlets was produced, so the methods described will allow mass propagation.     

Phenotypic variation during micropropagation of the chimeralRhododendron ‘President Roosevelt’

The putative periclinal chimeraRhododendron xlimbatum President Roosevelt was used to study the origin of shoots in vitro. Genotypic segregation readily occurred in vitro. Numerous phenotypes were observed, although most shoots were either entirely green or maintained the original variegation pattern. Derivatives of the third apical layer were rarely involved in shoot formation. A reversed chimeral form was isolated. Adventitious shoots were usually miniaturized and rapidly proliferating, but axillary shoots had thicker stems, larger leaves and proliferated more slowly. Corolla tissue produced stunted, leafy shoots; no variegated shoots were produced from floret explants. In shoot tip cultures the addition of 40M 2iP without IBA resulted in the greatest number of shoots. Explant choice was the most critical factor for maintenance of foliar variegation.     

Factors influencing the growth of micropropagated shoots and in vitro flowering of gentian

About 70% of the shoots developed from nodal explants ofGentiana triflora flowered in vitroondouble strength WPM medium containing 3% (w/v) sucrose, 0.5mg/l BA after 12 weeks of culture in a growth room at 22°Cwith continuous illumination (PPFD=60molm^–2 s^–1). The influences oninvitro shoot development and flowering of several factors includingthe position of the explant, requirements for sucrose, cytokinin orGA₃, variations of pH and photosynthetic photon flux density (PPFD)were investigated. In vitro flowering but not shootdevelopment of G. triflora decreased notably withincreaseddistance from the apex of the shoot, indicating the presence of a floralgradient in the micropropagated shoots. Conversely, as little as 0.01mg l^–1 GA₃ in the medium promotedshootdevelopment but even up to 0.2 mg l^–1GA₃ did not induce in vitro flowering.Even though BA could substitute GA₃ for a high level of shootdevelopment, it also promoted a high level of in vitroflowering at the PPFD of 60 molm^–2 s^–1. Sucrose was required for shootdevelopment and flowering in vitro and higher levels ofPPFD could not compensate effectively for the omission of the sugar from themedium. In general, the effects of different concentrations of BA in the mediumor variations of pH on shoot development and flowering invitro were found to be influenced by PPFD. A novel observation isthat precocious flowering of micropropagated gentian shoots did not occur ifthey were first cultured for 5 weeks in the dark before transfer to the lightcondition.     

The influence of cation and gelling agent concentrations on vitrification of apple cultivars in vitro

Shoot tips of York and Vermont Spur Delicious apples (Malus domestica Borkh.) were cultured in vitro to test the influence of K⁺, Mg⁺⁺ and gelling agent concentrations on vitrification. These concentrations were 20.05, 14.05 and 8.05 mM K⁺, 1.5 and 3.0 mM Mg⁺⁺, 7.0 g/l Difco Bacto agar and 1.0, 1.5 and 2.0 g/l Gelrite. The lowest K⁺ level produced a higher percentage of vitrified shoots, affected tissue appearance, reduced shoot number and shoot elongation and apparently altered shoot metabolic activity. Gelrite consistently produced vitrified leaves and stems, even though media gelled with 1.5 g/l Gelrite presented the same apparent gel firmness as using 7 g/l Difco Bacto agar, which did not induce vitrification. Less shoot elongation, fewer total shoots, and more usable shoots of York were obtained on Bacto-agar, while similar but less noticeable effects were obtained with Vermont Spur Delicious. The results presented here show that vitrification can be studied in a standardized system in which the only change is substitution of one gelling agent for another.     

Electrophoretic protein patterns of three species ofPhytophthora associated with black pod disease of cocoa (Theobroma cacao L.)

When electrophoretic profiles of native proteins from vegetative mycelia ofPhytophthora palmivora, Phytophthora capsici and Phytophthora citrophthora causing black pod disease of cocoa in India were compared on a single Polyacrylamide gel, the isolates of same species were readily distinguished both qualitatively by visual similarity in banding patterns and quantitatively by calculating similarity coefficients. Similarity coefficients were generally much higher between isolates within a species than between isolates of different species. The dendrograms obtained after unweighted pair grouping with arithmetic averaging cluster analysis, revealed that all the isolates ofPhytophthora capsici were highly homogenous and formed a single cluster. The isolates ofPhytophthora citrophthora were resolved into two electrophoretic types which were clustered into two distinct sub groups.Phytophthora palmivora formed a separate group. Thus, the results reveal that polyacrylamide gel electrophoresis can be used successfully in distinguishing species and sub groups within a species ofPhytophthora encountered on cocoa. CPCRl contribution No. 914.     

Brassinolide influences the regeneration of adventitious shoots from cultured leaf discs of tobacco

When several concentrations of brassinolide (BL) were added to a shoot induction medium (SIM) that contained only BA, redifferentiation of adventitious shoots from tobacco leaf discs was unaffected at low BL levels (10^-10~10^-8 M), but was inhibited at higher concentrations. In comparison, when BL was applied without BA, only cell expansion occurred and no shoots formed. The determination time for shoot formation was shortened at low BL concentrations, but their formation was postponed (i.e., time was lengthened) at higher concentrations. Elongation of shoots incubated for 30 d was unaffected at low BL concentrations, but was inhibited as that amount increased.NTH1, a tobacco homeobox gene that is expressed in the central zone of the tobacco shoot apex, showed greater expression levels in the SIM over time, and its expression was stronger in media treated with low concentrations of BL compared with the SIM control at the same time point. Expression ofNTH1 was postponed at higher BL concentrations. In conclusion, at low concentrations, brassinolide has no effect on shoot formation. However, it inhibits their formation at high concentrations when cytokinin is included in the media.     

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).     

Regeneration of Picea omorika plants via organogenesis

Picea omorika plants were regenerated from embryo and seedling shoot tip cultures. Adventitious and axillary shoots were produced on 1/2 MS medium containing benzyladenine and kinetin. Benzyladenine was more effective in bud induction, whereas kinetin hastened shoot development. Excised shoots were elongated on 1/3 MS medium without growth regulators, multiplied with kinetin and rooted with or without indole-3-butyric acid.Abbreviations BA N⁶-benzyladenine - 2IP N ⁶-(²-isopenteny) adenine - NAA -naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid     

In vitro micropropagation of the macarronesian evergreen treePersea indica (L.) K. Spreng

Summary Shoot propagation ofPersea indica (L.) K. Spreng was achieved using seedling axillary buds cultured on MS (Murashige and Skoog, 1962) medium with 1 mg/l (2.8 μM) N⁶-benzyladenine (BA). Forty percent of the obtained shoots did not elongate, but showed bud proliferation, which was maximal (three axillary buds per shoot) at the end of the seventh subculture. Sixty percent of the shoots elongated, did not show bud proliferation, and formed calluses at their base. Successful rooting (84.6%) was achieved dipping the base of each elongated shoot in 3 g/l (16.11 mM) indolebutyric acid (IBA) for 1–2 s, and transferring to half strength MS medium without growth regulators. These shoots presented an acclimatization success of 100%. Results suggest that micropropagated elongated shoots ofP. indica can be adequately used in reforestation programs.     

Comparison of somatic and sexual incompatibility between Datura innoxia and Atropa belladonna

After protoplast fusion somatic hybrid calli were obtained by complementation selection between an albino mutant of Datura innoxia and the wildtype of Atropa belladonna (Krumbiegel and Schieder, 1979. Planta 145, 371–375). In the present study experiments are described concerning leaf and shoot induction on several media supplemented with different combinations and concentrations of hormones. Except for fleshy leaves and embryos, no well-formed shoot could be obtained. However, under standard culture conditions after one and a half years, one line produced numerous green shoots, showing a reduced number of chromosomes from Atropa belladonna. The loss of some chromosomes decreased the degree of somatic incompatibility. The additional appearance of shoots with albino sectors, of total albino shoots, and of green shoots showing a different phenotype, demonstrated that the elimination of the chromosomes occurred not only once, but several times. At least one shoot nearly stable in chromosome content and green subline could be obtained possessing only 6 chromosomes of Atropa belladonna and the original chromosome number of Datura innoxia. Experiments were carried out to test the feasibility of producing sexual hybrids through in vivo and in vitro methods by cross pollination. However, no embryos, seeds, or plantlets were obtained, thus demonstrating that protoplast fusion is the only possibility for obtaining hybrids between these two species.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlor-phenoxyacetic acid - IAA indoleacetic acid - NAA -naphtaleneacetic acid - SDS sodiumdodecylsulfate     

Fruit-set and seed weight variation inAnthyllis vulneraria subsp.vulgaris (Fabaceae)

Fruit-set and seed weight variation was studied in a population ofAnthyllis vulneraria subsp.vulgaris (Fabaceae) in northwestern Spain. The plants produce several shoots, each bearing two to four inflorescences that open one at a time from bottom to top. Fruit-set, seed-pod weight and seed weight were found to be significantly higher in proximal inflorescences than in distal inflorescences of the same shoot; mean seed weight was up to three times higher in proximal than in distal inflorescences. By contrast, none of the three variables varied significantly among plants or among shoots of the same plant. Similarly, none of the three variables differed significantly between early- and late-flowering plants, or between plants monitored in 1993 and in 1994. These results are compatible with the view that shoots function as semiautonomous units as regards resource allocation, and that within the shoot resources are preferentially allocated to proximal (= early-opening) inflorescences. In the plants studied, the ratio of seed-pod weight to seed weight was fairly constant, suggesting that the pod is important for seed success.     

A survey of the response of Rhododendron to in vitro culture

The responses of 7 genotypes of Rhodendron to culture conditions and their establishment as shoot cultures are described. The genotypes represent a broad genetic diversity in the genus. After sterilization and an acclimation period of 3 to 12 months, all the selections were established as shoot cultures on Woody Plant Medium (WPM) supplemented with N⁶(-²-isopenteny) adenine (2iP). Plants with strong episodic growth cycles required the longer acclimation periods. Utilizing shoots from these cultures, the response to a cytokinin series of 0 to 32 M 2iP or BAP (6-Benzylaminopurine) was analyzed. BAP proved toxic to all but the elipidote and lepidote rhododendrons (R. mucronulatum, R. x Boule de Neige, and R. x PJM); however, even with these selections, 2iP stimulated greater shoot multiplication rates. The optimum 2iP level for shoot multiplication varied little with the genotype and levels of 4 to 16 M generally proved optimal, depending on the specific selection. Adventitious shoot production was observed in 3 selections (R. canadense, R. x Boule de Neige and R. x PJM), but only at 2iP levels above 8 M. Shoot multiplication rates of 7 to 21 times were observed, depending on the selection. Using an average utilizable shoot production rate of 40 shoots per culture per 6 week subculture period, some 75,000 shoots can be generated per square meter of culture space per year. The harvested shoots (microcuttings) rooted readily out-of-culture and the resultant plants grew like seedlings.     

Micropropagation of Campanula isophylla Moretti

A method for micropropagation of Campanula isophylla Moretti is described. The method is based on division of the basal parts of shoot clusters into sections, each with four 3 mm stem stubs. Shoots from the shoot clusters are easy to root and give plants without apparent phenotypic aberrations. It is thus possible to propagate the stock and produce rooted plantlets in the same process. Basal sections of shoot clusters formed more shoots than shoot tips or single nodes. The medium used for propagation was MS with 4.4 M benzyladenine (BA). Addition of naphthaleneacetic acid or raising the concentration of BA did not improve the results significantly. As primary explants 2 mm stem segments with an axillary or apical bud were used; smaller explants often failed to grow. For rooting the concentration of macronutrients was reduced to one-half, and BA was omitted. The cultures received an irradiance of 20 mol m^-2 s^-1 fluorescent light; dry weight of shoots decreased if the irradiance was reduced. The method was used for propagation of 113 genotypes; shoot numbers and days to first root differed significantly among genotypes.     

Efficient shoot regeneration of Brassica campestris using cotyledon explants cultured in vitro

Summary A system has been developed for efficient regeneration of shoots from Brassica campestris in vitro. Using 4-day old cotyledons with petioles as expiants and a combination of BA and NAA in the regeneration media, up to 70% of expiants produced shoots after 2 weeks in culture. The optimal conditions for regeneration were found to include a BA concentration of 2mgL^–1 and NAA concentration of 1mgL^–1. Light intensity had a profound effect on regeneration potential. The use of silver ions as an inhibitor of ethylene action reduced regeneration rates in this system. Rooting occured simultaneously with shoot formation on these media and the resultant shoots could be rooted readily on minimal medium. The genotype dependency was investigated and indicated that this method would be widely applicable to B. campestris cultivars. Regeneration of one cultivar, a high erucic acid type (R-500), was inefficient in the system described here. Histological studies indicated the development of multiple shoot primordia from the petiolar cut ends of the expiants after the initiation of meristematic activity in the cells about 100m from the cut site within 2 days of culture initiation. The system described is compatible with previously reported Agrobacterium — mediated transformation protocols involving cotyledonary petioles.     

Enhanced shoot regeneration from Brassica campestris by silver nitrate

Summary The morphogenetic response of Brassica campestris genotype R500 to inhibitors of ethylene biosynthesis and action was investigated. A medium containing 1.0 mg.l^–1 NAA, 2.0 mg.l^–1 BAP, and 30 or 60 M AgNO₃ significantly enhanced both the percentage shoot regeneration and the number of shoots per cotyledon expiant. Although callus proliferation occurred on hypocotyl segments, no shoots were formed in response to AgNO₃ with expiants older than five days. Cotyledons older than six days formed shoots only with AgNO₃. Cobalt chloride at 20 and 30 M increased cotyledon shoot regeneration but was inferior to AgNO₃. Hypocotyl segments were unresponsive. Salicylic acid at 25 and 50 M prevented both shoot regeneration and callusing without any obvious toxic effects. Removal of expiants from AgNO₃ after 12 days did not alter the percentage of shoot regeneration but increased the number of shoots per expiant. This response was dependent on the level of BAP. Percentage shoot regeneration and number of shoots per cotyledon explant were not affected by removal of CoCl₂. These results indicate that the poor regenerative capacity of this genotype may be related to ethylene biosynthesis or metabolism.Abbreviations NAA Naphthalene Acetic Acid - BAP 6-Benzylamino Purine - MS Murashige and Skoog Medium     

Multiple shoot induction by benzyladenine and complete plant regeneration from seed explants of chickpea (Cicer arietinum L.)

The efficacy of benzyladenine (BA) to induce multiple shoots from seed explants of chickpea (Cicer arietinum L.) was assessed. Shoot differentiation was influenced by the type of seed explant, genotype and concentration of BA. Orientation of the explant also strongly influenced the shoot regeneration response. The optimum BA concentration for shoot/shoot bud regeneration was genotype dependent. Two types of BA-induced response were observed: (1) at less than 7.5 gm BA, direct shoot differentiation (2 to 4-cm-long shoots) was observed within 30 days; (2) at higher BA concentrations (75–100 m), shoot/shoot bud differentiation was achieved in 45–90 days. A high BA concentration inhibited subsequent rooting of shoots. Roots, however, could be easily induced on shoots derived from <12.5 m BA. Following transfer to soil, 80% of the regenerants developed into morphologically normal and fertile plants.Abbreviations BA Benzyladenine