Viruses detected in Ullucus tuberosus (Basellaceae) from Peru and Bolivia

Ullucus tuberosus (Basellaceae) plants from 12 locations in the Andean highlands of Peru and Bolivia contained complexes of either three or four viruses. Specimens from six sites in Peru contained a potexvirus, a tobamovirus, a potyvirus and a comovirus, but those from another location lacked the potexvirus. All samples from five sites in Bolivia lacked the tobamovirus. The potexvirus (PMV/U) is a strain of papaya mosaic virus differing slightly from the type strain (PMV/T) in inducing milder symptoms in some common hosts and failing to infect a few other species. It symptomlessly infected U. tuberosus, and infected 15 of 29 species from seven of nine other families. PMV/U showed a close serological relationship to PMV/T and to boussingaultia mosaic virus and a distant relationship to commelina virus X, but it is apparently unrelated to any of ten other potexviruses. The tobamovirus (TMV/U) induced symptomless or inconspicuous infection in U. tuberosus, and infected 21 of 30 species from six of eight other families. It showed a very distant serological relationship to some strains of ribgrass mosaic, tobacco mosaic and tomato mosaic viruses, but failed to react with antisera to cucumber green mottle mosaic, frangipani mosaic, odontoglossum ringspot and sunn-hemp mosaic viruses. The potyvirus, tentatively designated ullucus mosaic virus (UMV), alone in U. tuberosus induced leaf symptoms indistinguishable from the chlorotic mottling and distortion found in naturally infected plants. UMV infected 12 of 20 species from four other families, and was transmitted in the non-persistent manner by Myzus persicae. It showed a distant serological relationship to only two (bidens mottle and alstroemeria mosaic) of 25 members or possible members of the potyvirus group tested. Some hosts and properties of the comovirus are described in an accompanying paper. None of the four viruses infected potato (Solanum tuberosum) and, with the possible exception of UMV, they differed from viruses reported previously to infect three other vegetatively propagated Andean crops (Oxalis tuberosa, Arracacia xanthorrhiza and Tropaeolum tuberosum).     

Germination of the seeds of ulluco (Ullucus tuberosus,Basellaceae)

Seeds of eight clones of ulluco (Ullucus tuberosus) were collected in 1985–1986 in cultivation experiments in the Botanical Garden of the University of Turku, and their germination was studied in a greenhouse and a growth chamber. The number of tested seeds was 1177. The disinfected seeds were tested in 10 different treatments with different illumination rhythms, temperatures, duration of treatments, and media. Stratification, removal of the calyx and seed coat, and mechanical rupture of the seed coat were used in the treatments. The mean germination percentage was 2.04%, but only five seedlings were obtained. Germinated seeds were obtained in three of the treatments; they belonged to clones from Puno, Cusco, and Juliaca. The best result was obtained by treating the seeds with 0.1% gibberellic acid, but the best seedlings, from the Puno clone, developed in pure water. The shortest germination time was 60 d and the longest 650 d, even within the same clone. The best illumination for germination was 8 h light and 16 h dark, in temperatures of +20°C (day) and +6°C (night). The seeds were found to germinate even after a storage period of 18 mo. The seedlings varied in habit, in size of seedling leaves, and in colour of stem, which could be light green or reddish.     

In vitro conservation of enset under slow-growth conditions

Studies on in vitro storage of enset under slow-growth conditions were carried out to develop an efficient protocol for conservation of the genetic diversity of the crop. The response to different growth retardation treatments was examined using three enset clones collected from southwestern Ethiopia. In vitro cultures could be effectively maintained for 6 months at 15 °C and 18 °C on MS medium supplemented with 10 μM BAP, in the presence of mannitol at concentrations of 0, 1 or 2% as a growth retardant. Shoots were subsequently recovered and multiplied on MS medium supplemented with 10 and 20 μM BAP at 25 °C and rooted shoots were successfully transferred to the greenhouse. Incubation at the lower temperature (15 °C) and the presence of mannitol in the culture medium had a significantly positive effect on maintenance, measured by the number of recovered shoots after storage. This revised version was published online in June 2006 with corrections to the Cover Date.     

Morphological variation among clones of ulluco (Ullucus tuberosus,Basellaceae) collected in Southern Peru

Material of 229 clones of ulluco (Ullucus tuberosus), representing 10 accessions from markets in southern Peru and one from the wild in Bolivia, was analyzed with regard to morphological variation. The diploid chromosome number (2n = 24) was ascertained in 16 of the clones. Clones grown in different environments (e.g., long vs. short day) retained a more or less similar order of variation with respect to color of the tubers, stems, and leaves and the length/width ratio of the leaves. The shape of fully developed tubers also showed stable variation. Within a single experiment several other vegetative characters showed significant differences between accessions. Furthermore, many flower and inflorescence characters showed variation between accessions, some of them also indicating geographical variation between the Puno-Juliaca area and the Cuzco area. Considerable variation between the clones of an accession was evident, some accessions being clearly more variable than others. Our results show that ulluco is a crop plant that varies extensively even within a geographically limited area. Although repeatedly referred to in the literature as a purely asexual crop, ulluco was shown to be capable of sexual reproduction; this must have been and possibly still is an important source of genetic variation in the species.     

Callus-mediated and direct protocorm-like body formation of Bletilla striata and assessment of clonal fidelity using ISSR markers

Two efficient morphogenetic pathways for micropropagation of Bletilla striata (Thunb.) Reichb. f. have been established through the callus-mediated and direct formation of protocorm-like bodies (PLBs) from protocorms and shoot tips. Green calli were induced from the basal surface of protocorms and the cut-end of shoot tips on Vacin and Went (VW) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) or α-naphthalene acetic acid (NAA) after 3–5 weeks, with the highest frequency of explants forming callus (48.0 %) from protocorms at 1.0 mg l^?1 2,4-D. The calli obtained from all plant growth regulator (PGR) treatments could proliferate and differentiate PLBs on the PGR-free medium. NAA and 2,4-D significantly enhanced the growth of callus. The fastest growth rate of callus was achieved at the combination of 1.0 mg l^?1 2,4-D and 1.0 mg l^?1 TDZ with 46.2-fold within 3 months. The regeneration of PLBs from callus was significantly improved by 6-benzyladenine (BA), and a mean number of 48.4 PLBs was produced from 100 mg calli at 1.0 mg l^?1 BA within 3 months. BA and thidiazuron (TDZ) promoted the direct formation of PLBs from explants. The highest frequency of direct PLBs formation (76.0 %) and the highest mean number of PLBs per explant (30.2) were observed in protocorms cultured with 0.5 mg l^?1 BA. Assessment of clonal fidelity by inter-simple sequence repeat (ISSR) markers revealed similarity ranges of 99.8–100.0 % between the regenerants and their mother plants and 99.5–100.0 % among the regenerants, which suggested the micropropagation protocols were genetically stable.     

Efficient micropropagation and assessment of genetic fidelity of Boerhaavia diffusa L- High trade medicinal plant

Physiology and Molecular Biology of Plants - Boerhaavia diffusa L is a medicinal herb with immense pharmaceutical significance. The plant is used by many herbalist, Ayurvedic and pharmaceutical...     

Plant regeneration from organogenic callus and assessment of clonal fidelity in Elephantopus scaber Linn., an ethnomedicinal herb

An efficient callus induction and plant regeneration system has been standardized for an ethnomedicinal plant, Elephantopus scaber Linn. Two explants i. e. seeds and leaf segments were used for callus induction. Murashige and Skoog (MS) medium supplemented with 5.0 μM 2, 4-dichlorophenoxy acetic acid (2, 4-D) and 0.5 μM kinetin (Kn) gave the optimum frequency (89 %) of callus induction from seed explant. The results showed that the highest response in terms of percent callus regenerating (91 %) and number of shoots (56) per culture was recorded on MS medium supplemented with 6.0 μM N₆-benzylaminopurine (BA) and 1.5 μM α naphthalene acetic acid (NAA). The best rooting of regenerated shoots was obtained on half strength MS medium supplemented with 6.0 μM indole-3- butyric acid (IBA). On this medium, 100 % of the shoots produced roots with a mean number of 3.2 roots per shoot. The positive role of vesicular arbuscular mycorrhizae (VAM) along with potting mix has been well established in the present study. Of the various potting mix employed for plant acclimatization, the highest response of 100 % plant survival was noticed when autoclaved garden soil, sand (2:1) and VAM was utilized as potting mix. Inter-simple sequence repeats (ISSR) were used to establish the clonal fidelity of regenerated plantlets and the banding profiles from callus derived plants were monomorphic and similar to those of mother plant, thus ascertaining the true-to-type nature of these plants.     

TDZ-induced axillary shoot proliferation of Rhododendron mucronulatum Turcz and assessment of clonal fidelity using DNA-based markers and flow cytometry

In Vitro Cellular & Developmental Biology - Plant - In order to induce in vitro axillary shoot proliferation from single-node explants of Rhododendron mucronulatum Turcz., two techniques of...     

In vitro culture of Capparis decidua and assessment of clonal fidelity of the regenerated plants

A protocol for in vitro multiplication of Capparis decidua (Forsk.) Edgew. has been developed from cultured leaves procured from multiplying axillary shoots on the cultured nodal explants. The highest efficiency of shoot formation was observed on Murashige and Skoog (MS) medium containing 2 mg dm⁻³ benzyladenine (BA) and 0.5 mg dm⁻³ 1-naphthaleneacetic acid. The regenerated shoots were transferred to MS medium containing 3 mg dm⁻³ BA for growth and proliferation. Shoots above 2 cm in length were transferred to MS medium supplemented with 1 mg dm-3 indole-3-butyric acid plus 0.5 mg dm⁻³ indole-3-acetic acid for root induction. No variation was detected among the micropropagated plants by randomly amplified polymorphic DNA (RAPD) markers.     

An efficient micropropagation protocol for Citrus jambhiri Lush. and assessment of clonal fidelity employing anatomical studies and RAPD markers

Citrus jambhiri (rough lemon) is considered a major rootstock source for a number of Citrus species. A simple method for micropropagation from nodal segments is reported. Nodal segments of C. jambhiri were inoculated on MS medium supplemented with different concentrations and combinations of 6-benzylaminopurine (BAP), kinetin, and N6-(2-isopentenyl) adenosine (2iP). Maximum multiple shoot regeneration response (75?%) was observed with BAP at 3?mg?l^?1. Shoots were multiplied for 30?d on fresh medium with similar composition. A total of 67?% of the cultures showed multiplication with the optimum number of shoots (4.02) and height of shoots (1.81?cm) with BAP (3?mg?l^?1) alone. Maximum rooting response (87?%) was observed with naphthaleneacetic acid at 0.5?mg?l^?1. Transverse sections of shoot stems obtained in vivo (sampled from seedlings) and in vitro (regenerated from nodal segments), showed similar anatomies. Randomly amplified polymorphic DNA analysis confirmed that all the regenerated plants were genetically identical to their donor plant, suggesting absence of detectable genetic variation in the regenerated plantlets.     

In vitro propagation and assessment of clonal fidelity of Nepenthes khasiana Hook. f.: a medicinal insectivorous plant of India

An efficient in vitro protocol for large-scale multiplication of Nepenthes khasiana, a threatened insectivorous plant of India, has been developed from nodal stem segments. The highest shoot proliferation of 19.16 ± 0.23 shoots/explant was recorded in half-strength Murashige and Skoog (MS) medium supplemented with 2.5 mg/l kinetin, 2.0 mg/l 6-benzyl aminopurine, 3 % sucrose and 0.8 % agar. The best rooting was achieved in half-strength MS medium supplemented with 2.0 mg/l α-naphthalene acetic acid with an average of 9.04 ± 0.46 roots/shoot. The plantlets were successfully transferred to the greenhouse with survival rate of 92 %, exhibiting normal development. Cytological and random amplified polymorphic DNA (RAPD) analyses were carried out to assess the genetic integrity of the regenerated plantlets. Cytological analysis revealed no change in chromosome number with cells studied showing 2n = 80. Of the 80 primers screened for RAPD analysis, 14 primers resulted in clear and scorable bands. A total of 72 amplification products were obtained out of which only 4.1 % bands were polymorphic. Cluster analysis of the RAPD profile revealed an average similarity coefficient ranging from 0.98 to 1.0, thus suggesting genetic stability in the micropropagated plants of N. khasiana.     

Regeneration of <Emphasis Type="Italic">Clivia miniata</Emphasis> and assessment of clonal fidelity of plantlets

An efficient in vitro micropropagation system for Clivia miniata Regel was developed using basal tissues of young petals and young ovaries as explants. For callus induction, explants were incubated on Murashige and Skoog (MS) medium containing either 2.22 μM 6-benzyladenine (BA) and 4.52 μM 2,4-dichlorophenoxyacetic acid (2,4-D) or 4.44 μM BA, 5.37 μM α-naphthaleneacetic acid (NAA), and 9.05 μM 2,4-D. Moreover, callus was induced from young ovaries when these were incubated on MS medium containing 8.88 μM BA, 10.74 μM NAA, and 9.05 or 18.10 μM 2,4-D. Subsequently, callus was transferred to MS medium supplemented with kinetin (KT) and NAA for shoot organogenesis. Frequency of shoot regeneration from petal-derived callus was highest when callus was transferred to medium containing 2.69 μM NAA with either 9.29 or 13.94 μM KT. Shoot regeneration frequency from ovary-derived callus was highest when this callus was transferred to medium containing 9.29 μM KT and 10.74 μM NAA. Overall, different explant types exhibited different organogenic capacities wherein, young petals had higher shoot regeneration frequencies than young ovaries. The highest rooting frequency (98.25 ± 3.04%) was obtained when shoots were transferred to half-strength MS medium without plant growth regulators. Regenerated plantlets were transplanted to soil mix and acclimatized, yielding a 96.80% survival frequency. Only 0.6% of regenerated plantlets exhibited morphological changes. The diploid status (2n = 22) of regenerated plantlets was determined using chromosome counts of root-tips. Moreover, inter-simple sequence repeats were used to assess the genetic fidelity of regenerated plantlets. Overall, regenerated plants shared 90.5–100.0% genetic similarities with mother plants and 89.0–100.0% similarities with each other.     

Nutritional evaluation of three underexploited andean tubers:Oxalis tuberosa (Oxalidaceae),Ullucus tuberosus (Basellaceae), andTropaeolum tuberosum (Tropaeolaceae)

Three species of edible tubers endemic to and domesticated in the Andes were studied for their nutritional value. Collected samples ofOxalis tuberosa, Ullucus tuberosus, andTropaeolum tuberosum show a high amount of variation in both percent protein and quality of essential amino acids. A protein difference of120% is present amongT. tuberosum cultivars and a protein difference of 300% is present among the three species. The data indicate that previous published Andean tuber crop food values may need revision. The introduction of "improved" crop varieties and less nutritious foodstuffs threatens the base ofcultivar diversity that has been selected by Andean agriculturists over centuries. This rapid erosion of Andean tuber diversity indicates the importance of identifying and conserving Andean tuber cul-tivars throughout the Andes.     

In vitro plant regeneration from cotyledonary nodes of Withania somnifera (L.) Dunal and assessment of clonal fidelity using RAPD and ISSR markers

An efficient large-scale clonal propagation protocol has been described for Withania somnifera (L.) Dunal, a valuable medicinal plant, using cotyledonary nodes derived from axenic seedlings. Murashige and Skoog’s (Physiol Plant 15:473–497, 1962) (MS) medium supplemented with 1.0 mg l^?1 N ⁶-benzyladenine (BA) was found to be optimum for production of multiple shoots (100 % shoot proliferation frequency and 16.93 shoots per explant). Successive shoot cultures were established by repeatedly sub-culturing the original cotyledonary node on a fresh medium after each harvest of newly formed shoots. Multiple shoot proliferation was also achieved from nodal segments derived from in vitro raised shoots on MS medium augmented with 1.0 mg l^?1 BA. Regenerated shoots were best rooted (95.2 %, 38.7 roots per shoot) in half-strength MS medium supplemented with 1.0 mg l^?1 indole-3-butyric acid. The plantlets were successfully acclimated and established in soil. Random amplified polymorphic DNA and inter-simple sequence repeats analysis revealed a homogeneous amplification profile for all micropropagated plants analyzed validating the genetic fidelity of the in vitro regenerated plants.     

Molecular analysis of micropropagated neem plants using aflp markers for ascertaining clonal fidelity

Summary The importance of neem (Azadirachta indica A. Juss.) as a medicinal tree species has been acknowledged worldwide. Superior trees with desired traits such as high azadirachtin content have been identified and micropropagated. Somaclonal variants that may arise in vitro, however, pose limitations to large-scale micropropagation. It is, therefore, imperative to establish genetic uniformity of such plantlets by ensuring strict quality checks at various stages of in vitro culture. This is the first study that evaluates the applicability of amplified fragment length polymorphism (AFLP) markers in establishing clonal fidelity of tissue culture(TC)-raised neem plants. Seven AFLP primer combinations generated a total of 334 amplified fragments across the mother plant, TC progenies, and other neem accessions that were included as controls. Two hundred and thirty-nine amplified fragments were monomorphic across the mother tree and its TC progenies. No extra band was detected in the TC plantlets that was absent in the mother tree, indicating that the TC plantlets regenerated through nodal explants are indeed true-to-type. Ninety-five AFLP fragments were detected in the controls, which allowed their discrimination from the elite mother tree and its TC progenies. Similarity matrix based on Jaccard's coefficient revealed that the pair-wise value between the mother tree and its TC plantlets was ‘1’, indicating perfect similarity. Phenetic dendrogram based on UPGMA (unweighted pair group method of arithmetic averages) analysis further confirmed the true-to-type nature of TC progenies, since a tie was observed between the mother tree and its TC plantlets. On the contrary, the control neem accessions were distinct from the mother and its TC progenies. AFLP markers proved to be an ideal tool for routine analysis and certification of genetic fidelity of micropropagated plants prior to commercialization, especially in tree species because of their long generation time.     

Assessment of genetic fidelity of encapsulated microshoots of <Emphasis Type="Italic">Picrorhiza kurrooa</Emphasis>

In vitro grown microshoots of Picrrhiza kurrooa were encapsulated in the alginate beads. Regrowth of encapsulated microshoots, using alginate encapsulation, of P. kurrooa reached 89.33% following 3 months of storage. Amongst developing plantlets, 42.66% exhibited formation of multiple shoots at the onset of regrowth and 21.43% demonstrated simultaneous formation of shoots and roots. Healthy root formation was observed in plantlets following 2 weeks of their transfer to half-strength Murashige and Skoog medium containing 1 μM α-naphthalene acetic acid. Plants were transplanted to the greenhouse in three batches with 95% frequency of survival. The genetic fidelity of P. kurrooa plants growing out after storage in encapsulated form was ascertained by random amplified polymorphic DNA (RAPD) analysis. Molecular analysis of randomly selected plants from each batch was conducted using 45 random decamer primers. Of 45 primes tested, 14 produced scorable amplified products. Total 68 bands were observed amongst them 7.35% bands were polymorphic. Cluster analysis of the RAPD profile revealed an average similarity coefficient of 0.966 thus confirming genetic stability of plants derived from encapsulated microshoots following 3 months of storage.     

Improved in vitro propagation,solasodine accumulation and assessment of clonal fidelity in regenerants of Solanum trilobatum L. by flow cytometry and SPAR methods

An efficient protocol for the development of genetically uniform clones of a valuable medicinal plant Solanum trilobatum L. has been established. An optimal shoot regeneration response was observed in a modified Murashige and Skoog medium (M-MS) containing 25 mM ammonium nitrate, 2 mg l^?1 6-benzyl adenine and 0.1 mg l^?1 indole-3-acetic acid using in vitro derived node and shoot tip explants. Consequently, the multiple shoot buds were elongated in MS medium supplemented with 0.5 mg l^?1 Gibberellic acid. The in vitro regenerated shoots were rooted best in MS medium containing 1.5 mg l^?1 indole-3-butyric acid and successfully acclimatized in the field. The single primer amplification reaction (SPAR) approach, including random amplified polymorphic DNA, inter simple sequence repeats and directed amplification of minisatellite DNA regions markers did not identify any genetic polymorphism among in vitro regenerants. Similarly flow cytometry analysis illustrated that the DNA content and genome size of micropropagated plants were equivalent to that of intact plants from field. In addition, the accumulation of solasodine in micropropagated plants was confirmed by thin layer chromatography and further quantified by high performance liquid chromatography analysis as 2.47 mg g^?1 DW which is comparable to field grown plants. Thus the protocol can be effectively exploited for commercial propagation of this species to obtain solasodine and also in genetic transformation studies.     

Synthetic seed technology for short term conservation of medicinal orchid Dendrobium densiflorum Lindl. Ex Wall and assessment of genetic fidelity of regenerants

Artificial seeds were obtained through encapsulation of protocorm-like bodies (PLBs) of Dendrobium densiflorum in calcium alginate beads. This paper demonstrates the alginate-encapsulation and conversion (complete plantlet regeneration) from PLBs, the effect of storage conditions (at different temperature; 4, 8, 16 °C, RT and duration; 15, 30, 45, 60, 75, 90 days) on viability of encapsulated plant materials as well as the assessment of genetic fidelity of the regenerants. Individual PLBs were encapsulated in calcium alginate beads for mass propagation, short-term storage and germplasm sharing. The superior gel matrix for encapsulation was obtained using 3 % sodium alginate and 100 mM calcium chloride (CaCl₂·2H₂O). The highest percentage of conversion (100 %) of encapsulated PLBs (capsules) was obtained on MS2 medium (MS medium + 2 mg/l BAP). Capsules were successfully stored till 60 days at 8 °C with conversion frequency of 95.5 %. Plantlets regenerated from encapsulated beads were acclimatized successfully with 95 % survival rate. A total of 40 primers were screened, out of which 10 primers successfully generated 39 scorable bands, ranging from 0.2 to 1.3 kb amplicons. The uniform RAPD banding profile among the plantlets derived from encapsulated PLBs following 60 days of storage confirmed genetic fidelity.