Recovering polyploid papaya in vitro regenerants as screened by flow cytometry

Several protocols have been proposed for in vitro propagation of papaya, either based on somatic embryogenesis or shoot organogenesis. It is well-known that tissue culture-based approaches are frequently associated with somaclonal variation. Whether on the one hand this phenomenon can preclude further stages of in vitro culture, on the other hand it can generate useful genetic variability for crop improvement. However, somaclonal variation analyses are limited in papaya tissue culture. The DNA ploidy level of 250 papaya somatic embryogenesis-derived plantlets from immature zygotic embryos was analyzed by flow cytometry. In vitro-grown and greenhouse seed-derived plantlets were used as diploid standards. Flow cytometry unambiguously evidenced euploid (diploid, mixoploid, triploid and tetraploid) and aneuploid papaya plantlets, indicating that in vitro culture conditions can lead the occurrence of somaclonal variation. Additionally, the two subsequent flow cytometry analyses showed that the DNA ploidy level remained stable in all cloned papaya plantlets during the successive subcultures in the multiplication medium.     

In vitro induction of tetraploids in cassava variety ‘Xinxuan 048’ using colchicine

Polyploids have great breeding value as they could have higher vegetative yields, higher qualities and greater tolerances against biotic and abiotic stresses. This research is focusing on in vitro colchicine-induced tetraploids in cassava. The survival rate of explants decreased with the increase of colchicine concentrations. Based on the survival rate, the treatment with 0.05 g l^?1 colchicine for 2 days was the best protocol for tetraploid induction in the cassava variety ‘Xinxuan 048’. The determination of ploidy levels showed that 28 autotetraploidy and 12 mixoploidy plantlets were obtained from 44 plantlets. Thus, 90.9% of the plants were variants. Significant differences in morphology and anatomy were found between the diploid and tetraploid plants. Tetraploid plants showed better photosynthetic capacities than the original diploid plants. The tetraploid cassava regenerated herein will enrich the germplasm spectrum and open a new arena for breeding novel triploids with elite cultivars by interploid crossing in the future.     

A protocol for fertility restoration of F<Subscript>1</Subscript> hybrid derived from <Emphasis Type="Italic">Lilium</Emphasis> × <Emphasis Type="Italic">formolongi</Emphasis> ‘Raizan 3’ × Oriental hybrid ‘Sorbonne’

The present study was an attempt to develop an in vitro colchicine chromosome doubling protocol to restore the fertility of an F₁ interspecific hybrid in Lilium. Basal scale segments of Lilium?×?formolongi?×?Oriental hybrid (FO) bulblets were pre-cultured for three durations (6, 15 and 25 days) and soaked in three colchicine concentrations (0.00, 1.25 and 2.50 mM) for 18, 24 and 36 h. To separate mixoploids, three cycles of adventitious bud induction were performed. The ploidy levels of the surviving plantlets were detected by flow cytometry at 30–31 weeks after induction and were confirmed by chromosome counts. The results indicated that the pre-culture duration, colchicine concentration and exposure time all had significant impacts on the tetraploid induction rate. The preferred procedure was to pre-culture the segments for 6 days and then treat them with 1.25 mM colchicine for 24 h. The morphological traits of rosette leaves were significantly different between the tetraploid and diploid plants. The adult tetraploid plants had considerably longer and wider leaves, larger flowers, and delayed flowering time (8 days later) than did diploid plants. Pollen viability tests and backcross trials of FO hybrids demonstrated fertility restoration at the tetraploid level. This protocol provides a feasible method for inducing fertile tetraploid FO hybrids for further breeding.     

Assessment of Genetic Stability in Three Generations of In Vitro Propagated <Emphasis Type="Italic">Jatropha curcas</Emphasis> L. Plantlets Using ISSR Markers

Tissue culture has been widely employed in Jatropha curcas L. for the clonal multiplication of superior genotypes. However, the evaluation of genetic stability is necessary to detect somaclonal variants. In this context, the present aim was to evaluate the genetic stability of J. curcas plantlets, obtained via indirect organogenesis, by means of ISSR markers. To supply the explant sources for in vitro propagation, the first generation of plants was produced from in vitro germination of J. curcas seeds. Fragments of cotyledonary leaves were inoculated into medium supplemented with 1.5 mg L^?1 BAP and 0.05 mg L^?1 of IBA for induction of callogenesis. The resulting calli were transferred to bud induction medium. Subsequently, the buds were cultured in medium for elongation, giving rise to the second generation of plants. These plants provided new buds, which were excised and subcultured in elongation medium, yielding a third generation of plants. To evaluate genetic stability in three plant generations, twelve ISSR primers were used, resulting in 124 bands showing 41.93 % of polymorphism. Increase was observed in the level of somaclonal variation (SV) over the generations. The present study reports, for the first time, the analysis of genetic stability in J. curcas plantlets regenerated via indirect organogenesis by means of ISSR markers. The results suggest that the indirect route is associated to higher levels of genetic instability, which also increased with successive subcultures. The ISSR markers were efficient in detecting SV, and the generated genetic variability may be useful for breeding programs.     

In vitro regeneration ability of diploid and autotetraploid plants of Cichorium intybus L.

Polyploidy has played a significant role in the evolutionary history of plants and is a valuable tool for obtaining useful characteristics. Because of the novelty of polyploids, comparison of their in vitro culture responses with diploids would be notable. In this study, leaf explants from diploid, autotetraploid and mixoploid plants of Cichorium intybus L. were cultured in vitro on the similar media and under same conditions. The ploidy level of the obtained calluses and regenerants were determined by flow cytometry analysis. The callogenic response of leaf explants cultured on the callus induction medium did not depend on the ploidy level of their parental plants. According to the flow cytometry analysis, the increased ploidy levels (4x) and (8x) were observed in the callus cultures with diploid and tetraploid origin, respectively. A considerable difference was observed between the ploidy level of mixoploid plants and their calluses, indicating the dominance of diploid cells in the callus tissue. The results showed that polyploidy led to the loss of organogenic potential as the tetraploid origin calluses failed to regenerate, while the diploid origin calluses successfully regenerated to whole plants.     

In vitro tetraploid induction and generation of tetraploids from mixoploids in hop (Humulus lupulus L.)

This paper describes an efficient colchicine-mediated technique for the in vitro induction of hop tetraploids and its confirmation by flow cytometry. A window of conditions generated a high percentage (>20%) of tetraploid induction, with the highest induction (25.6%) achieved with 0.05% colchicine for 48 h. Colchicine-induced tetraploids remained stable after 6 months in soil. Leaf characteristics of diploid and tetraploid hops were compared, and it was determined that stomatal length and width are suitable parameters for identifying putative hop tetraploids. As well as generating tetraploids, this technique generates mixoploid hops. Calli, derived from mixoploid leaves, were induced to form shoot buds and shoots. Individual shoots were classed as diploid, mixoploid or tetraploid after screening by flow cytometry. This callus-based technique can be employed when a genome-doubling agent generates mixoploids but fails to generate tetraploids.     

In vitro induction of tetraploids in Arachis paraguariensis

In this study, an efficient procedure was established for successful induction of tetraploid Arachis paraguariensis by treating diploid explants with colchicine. Quartered-seed, callus and shoot-tips were treated with colchicine at concentrations of 0.05, 0.1, 0.2 and 0.5?% (w/v) for 4, 8, 16, 20 and 24?h before they were transferred unto modified Murashige and Skoog medium for either callus induction or shoot regeneration. Results showed that quartered-seed displayed the highest frequency of in vitro plantlet regeneration and tetraploid induction, as well as the lowest mortality rate. Flow cytometric analysis also confirmed that the induced tetraploids from quartered-seed were true-to-type. The 0.5?% colchicine treatment for 4 to 8?h gave the best results with 39 and 43?% of the explants yielding tetraploid plants, respectively. Two?months following transfer to ex vitro environment, morphological and growth characteristics of the induced tetraploids were measured. Overall, increasing the ploidy level from 2× to 4× resulted in fewer stomata but more trichomes per unit leaf area. Tetraploid plants obtained in this study should expand the genetic base of Arachis, and can also be used in overcoming the existing hybridization barriers that may be due to ploidy differences within the genus Arachis.     

In vitro induction of polyploidy in Centella asiatica (L.) Urban

Mutational breeding was conducted using in vitro-grown shoot-tips of Centella asiatica (L.) Urban treated with colchicine (0.025–0.400% for 12–36 h) to induce polyploidy. Treated shoot-tips were grown on Murashige and Skoog (MS) medium supplemented with 4.54 μM thidiazuron (TDZ), and regenerated plantlets were acclimatized and transferred to soil. Two months following transfer to ex vitro conditions, ploidy levels of regenerated plants were determined by flow cytometry and by determining chromosome counts. Treating shoot-tips with colchicine concentrations ranging from 0.050–0.200% for 12–24 h promoted induction of tetraploids. Morphological and growth characteristics and the triterpenoid contents of the polyploids were also measured. Tetraploid plants demonstrated significantly longer stomata and a higher stomatal index compared to those of the diploid control plants. Furthermore, a positive trend in both biomass and triterpenoid production was obtained with the tetraploid and mixoploid plants of C. asiatica.     

In vitro induction and identification of tetraploid plants of <Emphasis Type="Italic">Paulownia tomentosa</Emphasis>

Polyploidization is a major trend in plant evolution that has many advantages over diploid. In particular, the enlargement and lower fertility of polyploids are very attractive traits in forest tree breeding programs. We report here a system for the in vitro induction and identification of tetraploid plants of Paulownia tomentosa induced by colchicine treatment. Embryonic calluses derived from placentas were transferred to liquid Murashige and Skoog (MS) medium containing different concentrations of colchicine (0.01, 0.05, or 0.1%) and incubated for 24, 48, or 72 h on an orbital shaker at 110 rpm. The best result in terms of the production of tetraploid plantlets was obtained in the 48 h + 0.05% colchicine treatment, with more than 100 tetraploid plantlets being produced. The ploidy level of plantlets was verified by chromosome counts, flow cytometry, and morphology. The chromosome number of tetraploids was 2n = 4x = 80 and that of diploid plantlets was 2n = 2x = 40. The relative fluorescence intensity of tetraploids was twofold higher than that of diploids. The tetraploid and diploid plantlets differed significantly in leaf shape, with those of the former being round and those of the latter pentagonal. The mean length of the stomata was longer in tetraploid plants than diploid plants, and stomatal frequency was reduced with the increased ploidy level. The tetraploids had large floral organs that were easily distinguishable from those of diploid plants.     

Development of mycorrhized vitroplants of Jatropha curcas L. at different rooting stages

In this work, we attempted to assess the effects of inoculation of arbuscular mycorrhizal fungus (AMF), Glomus clarum, on the survival and development of micropropagated Jatropha curcas plantlets at different rooting stages. Elongated shoots (average 3?cm) of J. curcas, maintained for 0, 14, or 21?days on rooting medium in the presence or absence of 1?mg?L^?1 indole-3-butyric acid (IBA), were transferred to a sand:soil:vermiculite (1:1/2:1) (v:v:v) substrate. At the time of transplantation, the plantlets were either inoculated or not inoculated with G. clarum that had been monoaxenically produced in Ri-transformed carrot roots. After a 2-week acclimatization period, 100?% of the plants kept for 0 or 14?days in rooting medium survived. However, those that remained for 21?days in rooting medium displayed post-acclimatization survival rates of 93 and 89?% for plants inoculated and non-inoculated with G. clarum, respectively. Colonization rates ranged from 70 to 93?%, and the stimulatory effects of AMF were evidenced by increased phosphorus uptake by plants and increases in all evaluated growth parameters, except plant height. Plants that were not subjected to the rooting stage showed growth similar to or higher than those subjected to the rooting stage, regardless of the addition of IBA. It can be concluded that stimulatory effects of mycorrhizal fungi were observed, and that the inoculation of J. curcas proved effective during the initial period of the acclimatization phase.     

Variation of cell cycle duration within suspension cultures ofDaucus carota and its consequence for the induction of ploidy changes with colchicine

Summary Continuous exposure to colchicine was used to estimate the variation in cell cycle time between cells within suspension cultures ofDaucus carota. Observations were made of the pattern of disappearance of cells of the initially predominant ploidy levels in diploid and tetraploid cultures having markedly different aggregation patterns. Both cultures showed a similar range of cycle times, normally distributed about the culture mean. Shorter colchicine treatments, followed by regrowth in colchicine-free medium, showed that spread of cycle times in the diploid culture prevented uniform induction of tetraploidy, and that the resulting mixoploid suspensions showed a gradual reversion to diploidy during subsequent subculture.     

In vitro production and identification of autotetraploids of Scutellaria baicalensis

The roots of Scutellaria baicalensis are a major traditional Chinese medicine. We report research on induction, characteristics and chemical analysis of polyploid plants of S. baicalensis. Immersing calluses in 0.2% colchicine solution for 12 h prior to culture induced a high number of tetraploid plants. The induction rate reached as high as 40% of treated calluses. More than 50 lines of tetraploid plants were obtained. All tetraploid plants showed typical polyploidy characteristics. Twenty selected tetraploid lines were transferred to the field for determination of morphological characteristics and for chemical assays. Seven elite lines have been selected for further selection and breeding into new varieties for commercial production.     

Antimitotic and hormone effects on green double haploid plant production through anther culture of Mediterranean japonica rice

Rice double haploid (DH) plants are produced mainly through anther culture. In order to improve the anther culture protocol, microspores of two japonica rice genotypes (NRVC980385 and H28) were subjected to three growth regulator combinations and four colchicine treatments on induction medium. In addition, a post anther culture procedure using colchicine or oryzalin was tested to induce double haploid plantlets from haploid plantlets. A cold pre-treatment of microspores for 9 days at 10 °C increased callus induction 50-fold in the NRCV980385 genotype. For both genotypes, 2 mg L^?1 2,4-D and 1 mg L^?1 kinetin on colchicine-free induction medium gave the best culture responses. The culturability of both genotypes changed on colchicine-supplemented induction media. A high genotype dependency was recorded for callus induction, callus regenerating green plantlets and regeneration of green double haploid plantlets. Colchicine at 300 mg L^?1 for 48 h enhanced callus induction 100-fold in H28. Colchicine-supplemented media clearly improved green double haploid plantlet regeneration. We showed that the post-anther culture treatment of haploid plantlets at 500 mg L^?1 of colchicine permitted fertile double haploid plantlets to be generated. Finally, an enhanced medium-throughput flow cytometry protocol for rice was tested to analyse all the plantlets from anther and post anther culture.     

Manipulation of ploidy for kiwifruit breeding: in vitro chromosome doubling in diploid <Emphasis Type="Italic">Actinidia chinensis</Emphasis> Planch.

Tetraploid plants were induced by colchicine treatment of in vitro leaf petiole segment cultures of five diploid Actinidia chinensis Planch. genotypes, including the commercially important, yellow-fleshed cultivar ‘Hort16A’, three female selections with red-fleshed fruit and one male pollinizer. Petiole segments were incubated on a shoot regeneration medium for a period of 4 weeks, and subsequently microshoots were treated with 0.05 or 0.1% colchicine. About one-third of the regenerated shoots were tetraploid following 0.05% colchicine treatment, more than with 0.1% colchicine treatment. Similar rates of tetraploid induction were achieved with all the genotypes tested. The efficiency of induction of polyploidy depended on the interaction between the types of in vitro culture chosen and the concentration of colchicine used. There are no previous reports of colchicine being used so successfully to induce polyploidy in Actinidia.     

Rapid micropropagation of monopodial orchid hybrid (Aranda Wan Chark Kuan ‘Blue’?×?Vanda coerulea Grifft. ex. Lindl.) through direct induction of protocorm-like bodies from leaf segments

A competent protocol for accelerated plant regeneration system via direct induction of protocorm-like bodies (PLBs) from leaf of orchid hybrid Aranda Wan Chark Kuan ??Blue???×?Vanda coerulea Grifft. ex. Lindl. was developed for the first time to establish a basis for mass production. Using tissue culture technique, the conditions for PLB induction from leaf explants and conversion of PLBs into plantlets were investigated. Leaves were transferred to MS medium containing different types and concentrations of cytokinins (namely, N⁶-benzyladenine, 6-furfurylaminopurine, N-phenyl-N ^??-(1,2,3-thidiazol-5-yl)urea/TDZ and zeatin) for PLB induction. By means of exploring the effects of cytokinins, it was determined that the optimum PLB induction occurred on MS media supplemented with 1.5?mg?l^?1 TDZ; whereby accordingly, PLB induction (with a frequency of 94.8?%) was observed as early as 8?days of culture and an average of 25 PLBs was obtained from 1?cm² leaf segment after 40?days of culture. Variable pressure scanning electron microscopy indicated the different developmental stages of PLBs in detail. Light/stereo microscopic observation showed the maturation of PLBs and gradual formation of shoot and leaf primordia. More than 96?% conversion (with well-developed shoots and roots) was achieved within the next 30?days of culture, when well developed PLBs were transferred in MS medium supplemented with 1?mg?l^?1 BA, 0.5?mg?l^?1 IBA plus 60?mg?l^?1 adenine sulphate. After 60?days of transfer in plastic pots filled with sand and perlite (2:1; v/v) and with charcoal and coconut fibre (1:1; v/v), subsequently, 90?% well-acclimatized plantlets were recovered.     

In vitro tetraploidization for the augmentation of wedelolactone in <Emphasis Type="Italic">Sphagneticola calendulacea</Emphasis> (L.) Pruski

A practical and reliable method for in vitro tetraploidization of Sphagneticola calendulacea (L.) Pruski [synonym Wedelia chinensis (Osbeck) Merrill] has been established to enhance the production of wedelolactone. Shoot tip and nodal explants from in vitro-grown culture (2n?=?50) were exposed to the antimitotic chemical, i.e., colchicine, at various concentrations (0, 0.025, 0.05, 0.1, 0.3, and 0.5%; w/v) for 12, 24, 36, 48, and 60 h. The treated explants were then incubated and proliferated on Murashige and Skoog (MS) medium fortified with 0.2 mg l^?1 thidiazuron and 0.05 mg l^?1 naphthalene acetic acid, followed by root induction in 1.0 mg l^?1 indole-3 acetic acid enriched ½MS medium. Treatment of shoot tips with 0.05% colchicine for 24 h supported the maximum rate of survival (63.33%) of explants as well as tetraploid induction (42.93%). Morphological, stomatal, and cytological characteristics along with the secondary metabolite content of the in vitro tetraploids were compared to that of diploids. The recovered tetraploid plants possessed superior plant height, stem diameter, leaf size, root number, and increased length and width of stomata but decreased stomatal frequency. The tetraploid plants demonstrated twice the chromosome number (2n?=?4x?=?100) than the diploids as confirmed through cytology, spectrophotometry and flow cytometry. High-performance thin-layer chromatography showed a significant enhancement in the wedelolactone content of tetraploid plants (541.48 µg g^?1 of dried sample) in comparison to diploid plants (325.43 µg g^?1 of dried sample), signifying the prospective of this technique for the trade value improvement.     

Callus induction and shoot organogenesis from anther cultures of Curcuma attenuata Wall

Curcuma attenuata is a highly valued ornamental. This study provides the first report on C. attenuata shoot organogenesis and plant regeneration. Immature anthers derived from 5 to 7?cm long inflorescences were isolated and cultured on different variations of Murashige and Skoog (MS) media to induce callus and then shoot organogenesis. When the 2-mm long anthers in which microspores were at the uninucleate developmental stage were cultured in the dark on MS medium containing 13.6???M 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.3???M kinetin (KT) for 15?days and then transferred to 40???mol?m^?2?s^?1 fluorescent light for 30?days, the percentage callus induction reached 33.3?%. After callus was transferred to various differentiation media and cultured in the light, 33.1?% of all callus cultures could differentiate into adventitious shoots on MS medium supplemented with 22.0???M 6-benzyladenine (BA), 0.53???M ??-naphthaleneacetic acid (NAA) and 1.4???M thidiazuron (TDZ) after culturing for 60?days. Over 95?% of plantlets survived after transplanting plantlets into trays with a mixture of sand and perlite (2: 1) for 20?days. Chromosome number, determined from the root tips of young plantlets, indicated that all plantlets were diploid (2n?=?84).     

<Emphasis Type="Italic">In vitro</Emphasis> induction and identification of autotetraploids of <Emphasis Type="Italic">Dioscorea zingiberensis</Emphasis>

Dioscorea zingiberensis is an important medicinal plant and a source of diosgenin in China. We report research on the induction, characteristics, and chemical assays of polyploid plants of D. zingiberensis. Immersing calli in 0.3% colchicine solution for 16 h prior to culture induced a high number of autotetraploid plants. The induction rate reached as high as 36.7% of treated calli. More than 50 lines of autotetraploid plants were obtained. All tetraploid plants showed typical polyploidy characteristics. Twenty selected tetraploid lines were transferred to the field for determination of morphological characteristics and for chemical assays. Six elite lines have been selected for further selection and breeding into new varieties for commercial production.     

Efficient In vitro Plant Regeneration Protocol from Leaf Explant of Jatropha curcas L — A Promising Biofuel Plant

An efficient in vitro plant regeneration from leaf-disc culture of Jatropha curcas L has been established. Adventitious shoot buds along with callus were induced from leaves of 2-year-old J. curcas plants cultured on Murashige and Skoog’s (MS) medium supplemented with TDZ (2 μM) BAP (2 μM) and IBA (1 μM), wherein 63.3% leaf explants responded. The multiplication of shoots was achieved from the adventitious shoot buds after transferring them to shoot induction medium. The highest number of shoots (9.7/explant) was achieved after 6 weeks of culture on MS medium containing 3 μM of BAR The welldeveloped shoots were rooted on MS medium supplemented with IBA (1.5 μM) with the rooting frequency of 53.3%. Addition of phloroglucinol (200 μM) to the medium enhanced the frequency of rooting to 76.7%. Regenerated plantlets were successfully transferred to field after initial acclimatization.