The inheritance of organogenic response in melon

Previous studies have demonstrated variation in organogenic competence among plants within a population ofCucumis melo. In order to determine if leaf explant response is under genetic control, we investigated the distribution of the shoot regeneration frequency in F₁ and F₂ generations from parents representing extreme values forin vitro organogenic response. Results suggest a genetic model with two genes, partial dominance, independent segregation and similar effects for both genes.     

Characterization and classification of different genotypes in a population of Cucumis melo based on their ability to regenerate shoots from leaf explants

Variability of the in vitro organogenic response from leaf explants of a seed population of Cueumis melo L. ev. Cantaloupe Charentais was analyzed to detect genotypic variability among individual seeds for shoot regeneration. Variation in shoot regeneration frequency among plants from different seeds was compared to that found among clonal replicas of each original plant. Clonal propagation was performed using in vitro culture of apical and axillary buds of axenic plants to avoid somaclonal variation. Results demonstrate the existence of highly significant differences among plants from the seed population. Regeneration frequency of leaf explants derived from clonally propagated sets of a single plant can be described by a binomial distribution for each original plant coming from one seed. Using data from stochastic simulation, we studied the accuracy of different analyses to detect the presence of genotypic heterogeneity in a population. These analyses, together with our experimental design, allowed the separation of genotypes differing up to 5% in their regeneration ability. Results of this work should allow researchers to hypothesize about those genotypes that differ within a population and their frequency.Abbreviations IAA indole-3-acetic acid - K (Kinetin) 6-furfuryl-aminopurine     

New insights into the in vitro organogenesis process: the case of <Emphasis Type="Italic">Passiflora</Emphasis>

We present evidences that ultrastructural electron microscope findings are valuable ways to understand the in vitro regeneration process, in particular in the yellow passion fruit. Shoot-regeneration was induced in hypocotyl and leaf-derived explants using 4.44 μM BAP, and the entire organogenic process was analyzed using conventional histology, scanning and transmission electronic microscopy. Both direct and indirect regeneration modes were observed in hypocotyl explants, but only direct regeneration occurred in leaf-derived cultures. In the direct pathway from both explant types, meristemoids developed into globular structures, here called protuberances. The peripheral meristematic layers of the protuberances displayed ultrastructural characteristics indicative of a high metabolic activity, and only these cells originated shoots and leaf primordia, the latter being frequent when leaf explants were used. Moreover, the peripheral cells of the protuberances derived from leaf explants lost adhesion during the culture, diminishing the regeneration rates. We recommend the use of hypocotyls as a source of explant to obtain shoots as well as a genetic transformation system for the yellow passion fruit. However, the direct pathway is preferred because a type of amitosis occurred in the peripheral cells of hypocotyl-derived calli, which has the potential to result in genetic instability of the regenerating plants/tissue.     

Formation of shoots from leaf axils of Alstroemeria: The effect of the position on the stem

Direct shoot regeneration was induced from leaf explants of Alstroemeria. The explants contained a leaf blade and a small portion of stem node, which were cut from the erect shoots of in vitro multiplicated plantlets. The shoot regeneration capacity of the excised leaf explants was significantly related to the position of the explant on the stem. The youngest explant which was located closest to the shoot apex gave the highest response. A gradient response toward the shoot apex was observed in percentage of shoot regeneration and in the number of shoots per regenerating explant. Histological studies revealed that the shoots were initiated at the leaf axils. The origin of the adventitious buds was located at the epidermal layer of stem peripheral cells. This revised version was published online in June 2006 with corrections to the Cover Date.     

Age and orientation of the cotyledonary leaf explants determine the efficiency of de novo plant regeneration and Agrobacterium tumefaciens-mediated transformation in Jatropha curcas L.

Effects of age and orientation of the explant on callus induction and de novo shoot regeneration from cotyledonary leaf segments of Jatropha curcas were studied. The callus induction and shoot regeneration capacity of cotyledonary leaf segments were found significantly related to the age of the explants and their orientation in culture medium. The youngest explant, derived from the cotyledonary leaf of germinated seed induced the highest regeneration response as compared to one- and two-week-old explants. A gradient response with age of the explant was observed in percentage of callus induction, shoot regeneration from callus and the number of shoots per regenerating callus. The explants cultured with their abaxial side in medium showed significantly higher regeneration response. The youngest explant was found to be most amenable to Agrobacterium-mediated transformation as compared to older explants. The fact that callus induced from the edges of the explant followed by de novo shoot induction, and strong transient gus expression observed in the edges of the explant are significant for routine Agrobacterium-mediated transformation and generation of stable transgenic plants in J. curcas.     

A two-stage pretreatment of seedlings improves adventitious shoot regeneration in sugar beet (<Emphasis Type="Italic">Beta vulgaris</Emphasis> L.)

The effects of a two-stage pretreatment of seedlings on the subsequent shoot regeneration capacity were investigated. Pretreated seedlings were obtained by germinating seeds on three different germination media and then further culturing on six different growth media. Lamina and petiole explants of two sugar beet (Beta vulgaris L.) breeding lines were then excised from the pretreated seedlings and cultured on five different shoot regeneration media. In both breeding lines, petiole explants produced significantly more shoots than lamina explants with higher frequencies of organogenic capacities; petiole explants of the lines M1195 and ELK345 produced a mean of 2.1 and 2.7 shoots per explant while their lamina explants produced 1.5 and 2.2 shoots per explant, respectively. A genotypic variation was evident as the line ELK345 was more productive for shoot development from both types of explants. In overall comparisons of different germination, growth and regeneration media, germination medium was most effective when supplemented with 0.5 mg/l 6-benzyladenine (BA) while both growth and regeneration media were most productive when contained a combination of 0.25 mg/l BA and 0.10 mg/l indole-3-butyric acid (IBA). Of all the treatments tested, the highest mean number of shoots per explant (8.3 shoots) and frequency of organogenic explants (75.6%) were obtained on regeneration medium supplemented with 0.25 mg/l BA and 0.10 mg/l IBA when petiole explants of the line ELK345 were excised from the seedlings that had been germinated on medium containing 0.5 mg/l BA followed by further growth on medium containing 0.25 mg/l BA and 0.10 mg/l IBA.     

Factors influencing the regeneration capacity of oilseed rape and cauliflower in transformation experiments

The efficiency ofAgrobacterium-based transformation technique in oilseed rape and cauliflower was influenced by cultivar specificity, donor plant age and explant type. Marked differences in demands for plant hormone contents in the regeneration medium were recorded already among different types of nontransformed explants. The highest regeneration capacity was recorded with stem and leaf segments isolated from one-month-old aseptically grown plants. The regeneration was markedly species-dependent. Regeneration of transformed plants from stem segments and thin layers isolated from field-grown oilseed rape plants (at the most 2% of regenerating explants) and from oilseed rape hypocotyls (0.8% of regenerating explants) and cauliflower (1.2% of explant regenerated transformed shoots) was achieved after disarmedAgrobacterium treatment. Hypersensitive reaction of explants could be prevented by using prolongedin vitro precultivation and delayed application of the selective agent.     

Genetic analysis of leaf explant regenerability inSolanum chacoense

An F₁ population consisting of 51 genotypes, derived from two unresponsive parental lines ofSolanum chacoense Bitt., was examined for shoot regeneration from leaf explants. Fourteen genotypes failed to respond whereas, among the responsive genotypes, four produced multiple shoots on over 90% of the explants. Estimates of broad-sense heritability were high for both frequency of responsive explants (83%) and the number of shoots per responsive explant (82%). The segregation of the F₁ hybrid progeny was in agreement with the theoretical ratios of a genetic model for tissue culture responsiveness involving three unlinked genes. This study confirms earlier findings concerning the genetic control ofin vitro shoot regeneration from leaf explants inS. chacoense.     

Comparison of Benzyl Adenine Metabolism in Two Petunia hybrida Lines Differing in Shoot Organogenesis

The uptake and metabolism of the cytokinin benzyl adenine (BA) was compared in two lines of Petunia hybrida Vilm. differing in their shoot organogenic response. Leaf transfer experiments using shoot induction medium containing 4.4 micromolar BA showed that leaf explants from petunia line St40 required a shoot induction period of 6 to 10 days for commitment to shoot organogenesis; whereas leaf explants from petunia TLV1 required 12 to 28 days. The short induction period of petunia St40 and the higher organogenic response was positively associated with a threefold higher absorption of BA from the medium, an increased BA ribotide metabolite pool, the presence of BA within the explant during the shoot induction period, and the production of an unidentified metabolite C. However, the study of petunia TLV1 leaf explants showed that neither BA nor metabolite C are required during the shoot induction period for eventual shoot development. The longer shoot induction period of TLV1 was associated with low BA uptake during 24 days, a decreasing ribotide metabolite pool, the absence of benzyl adenosine triphosphate and metabolite C throughout the study, and the absence of BA within the explant during the shoot induction period. Differences in the shoot organogenic response of these related plant lines have been shown to be associated with differences in exogenous cytokinin uptake and the subsequent metabolism of that hormone.     

The role of cytokinins in shoot organogenesis in apple

Effective regeneration in vitro is a necessary precondition for the implementation of different biotechnological approaches in plant breeding. Numerous studies have reported on regeneration from apple somatic tissues, and organogenesis has been proved to be influenced by several factors including mother shoots (genotype, size, type, and age of explant), in vitro conditions (dark period, light intensity, and quality), and others (wounding, orientation of leaf explants). However, one of the most important factors before and during the regeneration process is the type and concentration of cytokinin applied. Thidiazuron and benzyladenine are the most frequently used cytokinins in the regeneration systems, but their efficiency depends on genotype and other factors. Other cytokinins (e.g., zeatin and kinetin) have also been tested in several experiments and they were found in general to be less active. The organogenic ability of explants can also be increased by a properly selected cytokinin pre-treatment. Cytokinins applied in the pre-treatments can influence the leaf structure, which in turn can alter the regeneration capacity of the leaf explant. Interactions between factors of pre-treatments (hormones, light, and culture conditions) and factors of the regeneration phase should be considered. This review brings into focus the role of different cytokinins during in vitro shoot development, discussing their effects on the histology of leaves developed in vitro, and how this affects the subsequent regeneration process.     

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     

Thidiazuron-induced high-frequency plant regeneration from leaf explants of Paulownia tomentosa mature trees

Attempts were made to study the effect of thidiazuron (TDZ) on adventitious shoot induction and plant development in Paulownia tomentosa explants derived from mature trees. Media with different concentrations of TDZ in combination with an auxin were used to induce adventitious shoot-buds in two explant types: basal leaf halves with the petiole attached (leaf explant) and intact petioles. Optimal shoot regeneration was obtained in leaf explants cultured on induction medium containing TDZ (22.7 or 27.3 μM) in combination with 2.9 μM indole-3-acetic acid (IAA) for 2 weeks, and subsequent culture in TDZ-free shoot development medium including 0.44 μM BA for a further 4-week period. The addition of IAA to the TDZ induction medium enhanced the shoot-forming capacity of explants. The caulogenic response varied significantly with the position of the explant along the shoot axis. The highest regeneration potential (85–87%) and shoot number (up to 17.6 shoots/explant) were obtained in leaf explants harvested from the most apical node exhibiting unfolded leaves (node 1). An analogous trend was also observed in intact petiole explants, although shoot regeneration ability was considerably lower, with values ranging from 15% for petioles isolated from node 1 to 5% for those of nodes 2 and 3. Shoot formation capacity was influenced by the genotype, with regeneration frequencies ranging from 50% to 70%. It was possible to root elongated shoots (20 mm) in basal medium without growth regulators; however, rooting frequency was significantly increased up to 90% by a 7-day treatment with 0.5 μM indole-3-butyric acid, regardless of the previous culture period in shoot development medium (4 or 8 weeks). Shoot quality of rooted plantlets was improved not only by IBA treatment but also by using material derived from the 4-week culture period. Regenerated plantlets were successfully acclimatized in the greenhouse 8 weeks after transplanting.     

Variation of phenotype,ploidy level,and organogenic potential of in vitro regenerated polyploids of <Emphasis Type="Italic">Pyrus communis</Emphasis>

A wide range of phenotypic variation was observed among neopolyploids obtained from the diploid pear cultivar ‘Fertility’ by in vitro colchicine treatment. The variant plantlets had alterations in leaf characteristics. Neopolyploids had significantly different ratios of leaf length to leaf width compared to the diploid control. Shoot regeneration from leaf explants and rooting ability from in vitro shoots of neopolyploids was examined. Regeneration frequencies of shoots from leaf explants of seven of the nine neopolyploids were significantly decreased compared to the diploid control. The organogenic potential of neopolyploids was highly genotype-dependent for both shoots and roots. Tetraploid clone 4x − 4 failed to regenerate shoots from leaf explants and the pentaploid clone 5x − 2 failed to root from in vitro shoots. The results suggest that polyploidization caused the decrease in or loss of in vitro organogenic potential. Regenerated shoots derived from neopolyploids showed different phenotypes, depending on the ploidy of the donor plant.     

Induction of adventitious shoots in vitro in Campanula carpatica

Efficient protocols have been developed to induce adventitious shoots in different types of explants of Campanula carpatica Jacq. More than five shoots per explant developed on hypocotyls of 5-week-old seedlings after 2 weeks of culture. Hypocotyls produced twice as many shoots as the cotyledons. TDZ proved to be about 6 times more efficient than BA. NAA had to be added to the regeneration medium to obtain the optimal balance of auxin and cytokinin to induce shoot regeneration. Significant differences were noted between different growth regulator concentrations in their effects on shoot organogenesis. BA induced double the number of callus clumps as TDZ. Incubation of explants in the dark produced about 6 shoots per explant while those in the light produced about 2 shoots per explant. Explants derived from 5-week-old seedlings were five times more regenerative compared to those derived from 15-week-old seedlings. Explants from cv. White Uniform were more organogenic than those from cv. Blue Clip. Root segments were also found to form shoots when treated with CPPU.     

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     

A shoot regeneration protocol effective on diverse genotypes of sunflower (Helianthus annuus L.)

Summary We describe a protocol, and several experiments that helped lead to its development, for sunflower regeneration. Important factors for sunflower regeneration were explant age, cytokinin type and concentration, basal medium, and explant source. We could not induce shoot regeneration from the explants derived from mature tissues including leaf, petiole, and stem. However, use of juvenile explants such as embryo meristem and primordial leaf tissues allowed routine regeneration of 17 different sunflower genotypes. High frequency of shoot regeneration was achieved with these explants taken from seedlings up to 5 d after germination. Explant age was less critical for embryo meristem explants than for primordial leaf tissues. Of the four basal media tested, MS and B5 media produced higher shoot-regeneration frequencies than did Anderson and woody plant media. The highest shoot-regeneration frequency was obtained with MS medium supplemented with 2 μM BA and without auxin. Addition of 1 μM naphthalene-acetic acid to the medium significantly reduced both the percentage of explants producing shoots and average number of shoots per explant. Regenerated shoots were grown to maturity in a greenhouse.     

Developmental and hormonal regulation of direct shoot organogenesis and somatic embryogenesis in sugarcane (Saccharum spp. interspecific hybrids) leaf culture

Rapid and efficient in vitro regeneration methods that minimise somaclonal variation are critical for the genetic transformation and mass propagation of commercial varieties. Using a transverse thin cell layer culture system, we have identified some of the developmental and physiological constraints that limit high-frequency regeneration in sugarcane leaf tissue. Tissue polarity and consequently the orientation of the explant in culture, size and developmental phase of explant, and auxin concentration play a significant role in determining the organogenic potential of leaf tissue in culture. Both adventitious shoot production and somatic embryogenesis occurred on the proximal cut surface of the explant, and a regeneration gradient, decreasing gradually from the basal to the distal end, exists in the leaf roll. Importantly, auxin, when added to the culture medium, reduced this spatial developmental constraint, as well as the effect of genotype on plant regeneration. Transverse sections (1-2 mm thick) obtained from young leaf spindle rolls and orienting explants with its distal end facing the medium (directly in contact with medium) are critical for maximum regeneration. Shoot regeneration was observed as early as 3 weeks on MS medium supplemented with alpha-naphthalenencetic acid (NAA) and 6-benzyladenine, while somatic embryogenesis or both adventitious shoot organogenesis and somatic embryogenesis occurred on medium with NAA and chlorophenoxyacetic acid. Twenty shoots or more could be generated from a single transverse section explant. These shoots regenerated roots and successfully established after transplanted to pots. Large numbers of plantlets can be regenerated directly and rapidly using this system. SmartSett, the registered name for this process and the plants produced, will have significant practical applications for the mass propagation of new cultivars and in genetic modification programs. The SmartSett system has already been used commercially to produce substantial numbers of plants of orange rust-resistant and new cultivars in Australia.     

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     

Adventitious shoot regeneration from leaf, stem and root explants of commercial cultivars of Gentiana

Several culture conditions were examined for promoting efficient plant regeneration from explants of Gentiana. Adventitious shoot regeneration from leaf explants of cv. WSP-3 was very superior on MS medium, compared to B5 medium, supplemented with four cytokinins (TDZ, 4PU-30, BA and zeatin). An auxin / cytokinin combination was required for regeneration. TDZ was the most effective cytokinin, while NAA was more effective than IAA or 2,4-D. Optimum conditions for regeneration from explants (leaf, stem and root) of cv. WSP-3, evaluated in terms of regeneration frequency and number of regenerated shoots per explant, were TDZ and NAA in combination, 5–10 mg/l and 0.1 mg/l for leaf and stem explants, and 10 mg/l and 1 mg/l for root explants, respectively. Application of these conditions to eight other commercial cultivars resulted in 30–100% regeneration from leaf explants. The number of chromosomes in each of ten regenerated plants of each cultivar was diploid, 2n=26. Shoots regenerated in vitro were rooted in phytohormone-free medium and transferred to soil.Abbreviations MS medium Murashige and Skoog's medium (Murashige and Skoog 1962) - B5 medium Gamborg B5 medium (Gamborg et al. 1968) - BA 6-benzylaminopurine - TDZ N-phenyl-N'-1,2,3-thiadiazol-5-yl urea - 4PU-30 N-(2-chloro-4-pyridyl)-N'-phenylurea - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid     

In vitro shoot regeneration from cotyledons of immature ovules ofImpatiens platypetala Lindl.

Summary An efficient and reproducible protocol has been developed for in vitro shoot regeneration from cotyledonary explants derived by germinating immature ovules ofImpatiens platypetala Lindl. &#x2018;TR6-27-2&#x2019;. Cotyledonary explants were cultured on a modified Murashige and Skoog (MS) agar-solidified medium containing 7.5g &#x00B7; liter^&#x2212;1 sucrose, 22.2&#x00B5; M N⁶-benzyladenine (BA), and 0.54&#x00B5;M &#x03B1;-naphthaleneacetic acid (NAA). The induction of organogenic tissues occurred after 6 to 8 wk in culture. Exogenous auxin and cytokinin were essential for the induction of organogenic tissues and survival of explants, and BA was most effective for the induction of organogenic tissues, compared with other cytokinins tested. The addition of glutamine (500 mg &#x00B7; liter^&#x2212;1) was also important for growth of organogenic tissues after induction and for reducing explant death during culture. The induction of organogenic tissue was also influenced by the type of cotyledon cultured and the age of the donor seedlings. On average, eight shoots per explant were induced from organogenic tissues larger than 0.5 cm in diameter 6 to 8 wk after transfer to a modified MS agar-solidified medium without NAA and BA reduced to 4.44&#x00B5;M. Shoots longer than 0.5 cm in length were successfully rooted 2 to 4 wk after transfer to a basal MS medium containing 30g &#x00B7; liter^&#x2212;1 sucrose.