Assessment Of Genetic Fidelity Research Articles

Micro-propagation of Hypericum gaitii Haines, an endangered medicinal plants : assessment of genetic fidelity

An efficient in vitro protocol was established for mass production of. Hypericum gaitii Haines, an important medicinal plant. Multiple shoots were induced from apical and axillary meristems on Murashige and Skoog medium supplemented with 1.0–3.0 mg/l 6- benzylaminopurine (BAP) or Kinetin and 3 % (w/v) sucrose. Inclusion of 0.5 mg/l 1-napthaleneacetic acid (NAA) in the culture medium to enhance the rate of shoot elongation and multiplication. The rate of multiplication was higher when the cultures were incubated under 16 h photoperiod rather than the continuous light. Rooting was readily achieved upon transferring the microshoots onto full strength MS basal semi-solid medium supplemented with 0.5–1.0 mg/l IBA after 15 days of culture. Medium having NAA along with BAP did not show the induction of rooting. Micro-propagated plantlets were acclimatized and successfully grown in soil mixture. About 50 % of micropropagated plantlets were hardened in the polyhouse and successfully established in the soil. Inter Simple Sequence Repeat DNA marker was used to detect the variability among the micropropagated plants developed through in vitro. The results showed that there was no polymorphism among the micropropagated plants. Further study is necessary to enhance the survivality rate of in vitro raised plantlets of Hypericum gaitii for commercialization.

Retraction Note to: Plant regeneration in Chlorophytum borivilianum Sant. et Fernand. from embryogenic callus and cell suspension culture and assessment of genetic fidelity of plants derived through somatic embryogenesis.

[This retracts the article DOI: 10.1007/s12298-012-0113-y.].

GERMPLASM CHARACTERIZATION, PROPAGATION AND MAINTENANCE OF HEALTH-PROMOTING WILD BERRIES AND MEDICINAL PLANT ROSEROOT (RHODIOLA ROSEA L.)

Berry crops include, but are not limited to the members of the genera: Fragaria (strawberry; Rosaceae), Rubus (brambles: raspberry and blackberry; Rosaceae), Vaccinium (blueberry, cranberry and lingonberry; Ericaceae) and Ribes (currant and gooseberry; Grossulariaceae). Berry fruits contain relatively high levels of vitamin C, cellulose, and pectin, and produce anthocyanins which have important therapeutic values, including antitumor, antiulcer, antioxidant and anti-inflammatory activities. Blueberry, cranberry and lingonberry are three Vaccinium fruit crops of economic importance which have been domesticated in the twentieth century. Cloudberry (Rubus chamaemorus L., family Rosaceae) is a less know small fruit of medicinal importance. The roseroot (Rhodiola rosea L.), also called golden root or arctic root, is used as an adaptogen in herbal medicine and is valued for its ability to enhance human resistance to stress or fatigue and promote longevity. There is a pressing need to develop reliable methods for conserving, maintaining and identifying berry germplasm and for assessing genetic biodiversity for practical breeding purposes and proprietary-rights protection. The introduction of molecular biology techniques, such as DNA-based markers, allows direct comparison of different genetic material independent of environmental influences. This paper presents the progress in-depth of various aspects of molecular diversity analyses in wild berry species and cultivars collected from Canada, Europe and USA. Inter simple sequence repeat (ISSR), random amplified polymorphic DNA (RAPD), expressed sequence tag-polymerase chain reaction (EST-PCR) and EST-simple sequence repeat (SSR) markers detected a sufficient degree of polymorphism to differentiate among wild clones and cultivars, making these technologies valuable for cultivar identification and for the more efficient choice of parents in berry breeding programs. Tremendous progress in plant tissue culture, resulting in great advances in micropropagation, has also been occurred. Although automation of micropropagation in bioreactors has been advanced as a possible way of reducing propagation cost, optimal plant production depends upon better understanding of physiological and biochemical responses of plant to the signals of culture microenvironment and an optimization of specific physical and chemical culture conditions to control the morphogenesis of berry plants in liquid culture systems. The paper describes the progress in-depth of various aspects of molecular characterization of berry and roseroot germplasm, their propagation and maintenance, and on the employment of molecular markers for the assessment of genetic fidelity, uniformity, stability and trueness-to-type among donor plants and tissue culture regenerants.

Clonal in vitro multiplication of grey mangrove and assessment of genetic fidelity using single primer amplification reaction (SPAR) methods

An efficient protocol for clonal multiplication of an important mangrove, Avicennia marina, was developed through in vitro culture of nodal segments obtained from a mature plant. The nodal explant induced multiple shoots when cultured on the Murashige and Skoog (MS) basal medium supplemented with varying concentrations and combinations of 6-benzyladenine (BA) and α-naphthalene acetic acid. The highest response in terms of per cent regeneration (73%), average number of shoots/explant (3.25 ± 0.25) and maximum shoot length (5.2 ± 0.27 cm) was obtained on the MS medium supplemented with BA 5.0 μmol/L + NAA 1.0 μmol/L + 3 g/L activated charcoal after 8 weeks of culturing. The regenerated shoots were rooted well in the MS medium supplemented with 1.0 μmol/L indole-3-butyric acid with an average of 2.9 ± 0.24 roots per microshoot. The rooted plantlets were successfully transferred to pots containing normal garden soil with 70% survival rate. The genetic stability of the regenerated plants was evaluated using single primer amplification reaction (SPAR) methods viz., random amplified polymorphic DNA, directed amplification of minisatellite DNA and intersimple sequence repeat polymorphism. The SPAR analysis revealed monomorphic banding patterns in all in vitro regenerated plantlets of A. marina and similar with that of the mother tree confirming their genetic uniformity and clonal fidelity.

Micropropagation and assessment of genetic fidelity of Henckelia incana: an endemic and medicinal Gesneriad of South India.

Henckelia incana is an endemic medicinal plant used for the treatment of fever and skin allergy. In the present study shoot regeneration was evaluated on Murashige and Skoog's (MS) medium supplemented with auxins, Indole-3-acetic acid (IAA), Indole-3- butyric acid (IBA), 1-Naphthaleneacetic acid (NAA), 2, 4-Dichlorophenoxyacetic acid (2, 4-D) and cytokinins, 6-Benzylaminopurine (BAP) and Kinetin (Kn) at concentrations of 0.5, 1.0, 2.0, 3.0, 4.0 and 5.0mgl(-1). MS medium with IBA (18.08), NAA (17.83) and IAA (17.58) at 0.5mgl(-1) concentrations showed efficient regeneration. Regenerated shoots were rooted on half-strength MS medium with and without 0.5mgl(-1) IBA or NAA. The plantlets were successfully hardened in rooting trays (peat, vermiculite and sand) and transferred to field mileu. The genetic fidelity of in vitro raised plants was assessed by using three different single primer amplification reaction (SPAR) markers namely random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR) and direct amplification of mini-satellite DNA region (DAMD). The results consistently demonstrated true-to-true type propagation. This is the first report of in vitro propagation and establishment of true-to-true type genetic fidelity in H. incana.

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

Boerhaavia diffusa L is a medicinal herb with immense pharmaceutical significance. The plant is used by many herbalist, Ayurvedic and pharmaceutical industries for production biopharmaceuticals. It is among the 46 medicinal plant species in high trade sourced mainly from wastelands and generally found in temperate regions of the world. However, the commercial bulk of this plant shows genetic variations which are the main constraint to use this plant as medicinal ingredient and to obtain high value products of pharmaceutical interest from this plant. In this study, we have regenerated the plant of Boerhaavia diffusa L through nodal explants and evaluated genetic fidelity of the micropropagated plants of Boerhaavia diffusa L with the help of random amplified polymorphic DNA (RAPD) markers. The results obtained using RAPD showed monomorphic banding pattern revealing genetic stability among the mother plant and in vitro regenerated plants of Boerhaavia diffusa L.

Nutrient optimization for improved in vitro plant regeneration in Eclipta alba (L.) Hassk. and assessment of genetic fidelity using RAPD analysis

This study highlights the effect of different inorganic micronutrients like copper, cobalt, molybdenum, zinc, boron, iodine, iron and manganese in accelerating and amplifying in vitro shoot bud induction and proliferation of a medicinally important plant, Eclipta alba (L.) Hassk. Direct shoot bud induction was observed on MS fortified with Kn (2 mg/l). However, maximum number of shoots was achieved when GA3, 0.5 mg/l was added to induction medium along with 1?M copper sulphate (ten times the normal MS level). Optimization of nutrient level in the basal medium promoted maximum regeneration response from both shoot tips and nodal explants. Elongated shoots were rooted in MS supplemented with IBA, 1.0 mg/l. Healthy, green plantlets with well developed roots, flowered normally in the field. Genetic stability of micropropagated plantlets was evaluated using RAPD markers. The amplification products were monomorphic in micropropagated plantlets and similar to those of mother plant revealing the genetic uniformity of plantlets. The regeneration protocol is highly efficient and reproducible so would be useful for mass multiplication, ex situ conservation and genetic transformation of E. alba (L.) Hassk.Plant Tissue Cult. & Biotech. 24(2): 223-234, 2014 (December)

An Improved Micropropagation and Assessment of Genetic Fidelity in Multipurpose Medicinal Tree, Acacia auriculiformis

An efficient direct plant regeneration protocol has been developed for medicinal tree, Acacia auriculiformis that can be employed at a commercial scale. Bioactive properties of this tree attribute to spermicidal action and anti-HIV activity which has gained an increasing interest in world health sector. Among several media employed for present study, an utmost shoot regeneration frequency of 93 % with highest number 17.3 ± 0.61 of shoots per explant was obtained on MS medium supplemented with 2.0 mg l−1 KIN and 0.5 mg l−1 IAA. Inclusion of activated charcoal in media has effectively alleviated the browning problem of shoots. Maximum shoot length of 6.4 cm was attained on MS media supplemented with 0.25 mg l−1 GA3 followed by rooting of individual microshoots on half strength MS media supplemented with 0.1 mg l−1 IAA. After successful hardening, rooted microshoots were maintained under greenhouse conditions. Genetic stability of micropropagated plants of A. auriculiformis have also been established via randomly amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) markers. A total of 83 (39 RAPD and 44 ISSR) clear, distinct and reproducible amplicons were produced. The banding patterns and similarity coefficients obtained by both RAPD and ISSR analysis have revealed no remarkable differences between tissue culture raised plants and the mother plant. This is the first report that evaluates the use of genetic markers to establish genetic fidelity of micropropagated A. auriculiformis. This method can be applied for the rapid clonal multiplication and true-to-type production of plant for attaining the ever increasing demand in pharmaceutical industries.

Genetic fidelity assessment of in vitro-regenerated plants of Albizia julibrissin using SCoT and IRAP fingerprinting

A protocol was established for callus induction and plant regeneration of Albizia julibrissin Durazz., a multipurpose tree. Calli were induced on hypocotyl explants excised from 10- to 14-d-old in vitro seedlings cultured on Murashige and Skoog (MS) medium supplemented with α-naphthaleneacetic acid (NAA) alone or in combination with 6-benzylaminopurine (BA) or 6-furfurylaminopurine (kinetin). The highest frequency of organogenic callus (82.2 ± 3.6%) was obtained on MS medium with 10.8 μM NAA and 4.4 μM BA. Calli were then cultured on MS medium with BA or zeatin, singly or in combination, for shoot regeneration. Calli cultured on MS medium with 13.2 μM BA and 4.6 μM zeatin produced the highest frequency of adventitious shoot regeneration (75.3 ± 6.3%). Maximum rooting of shoots (73.3 ± 5%) was achieved using half-strength MS medium with 4.9 μM indole-3-butyric acid. The genetic fidelity of 12 plants acclimatized to the greenhouse was assessed based on analyses of start codon targeted (SCoT) polymorphism and inter-retrotransposon amplified polymorphism (IRAP). The 14 SCoT and 7 IRAP adapted primers produced 71 and 34 scoreable fragments, of which 33 (46%) and 12 (35%) were polymorphic, respectively. The in vitro-raised plants exhibited 0.129–0.438 genetic distance from the mother plant and 0.000–0.788 distance from one another according to the SCoT and IRAP analyses. Although the culture method described here may not be suitable for clonal propagation of elite genotypes, it can be used for conservation of this plant.

Global DNA Methylation Levels During the Development of Nodule Cluster Cultures and Assessment of Genetic Fidelity of In Vitro-Regenerated Pineapple Plants (Ananas comosus var. comosus)

Pineapple presents a significant portion of the global fruit market. Recently, a micropropagation protocol was established based on nodule cluster cultures (NC), which showed high regenerative efficiency. Aiming to expand the understanding of gene regulation in this protocol, global DNA methylation levels (GDML) and fresh mass increase ratio (FMIR) during NC multiplication and differentiation to microshoots were evaluated after 0, 9, 18, 27, and 36 days in culture. In addition, genetic fidelity of randomly chosen regenerated plants was analyzed by means of amplified fragment length polymorphism (AFLP), flow cytometric analysis of ploidy level, as well as observation of phenotypic features. An association between GDML and the density of new shoot apical meristems formed in cultures was observed, which was, in turn, related to FMIR and the age of each subculture. The analysis by means of AFLP and DNA ploidy level, as well as phenotypic observations indicated the homogeneity of regenerated plantlets.

Genetic fidelity assessment in micropropagated plants using cytogenetical analysis and heterochromatin distribution: a case study with Nepenthes khasiana Hook f.

Rapid clonal propagation of selected genotypes has been one of the most extensively exploited approaches of biotechnology. However, inclusion of somaclonal variations in tissue-culture-derived plants results in the production of undesirable plant off-types which limits its applications in tissue culture industry. Therefore, the most critical concern has been the maintenance of genetic uniformity of micropropagated plants. Assessment of genetic fidelity in tissue-culture-raised plants of three consecutive regenerations of Nepenthes khasiana has been successfully carried out using chromosome counts and heterochromatin distribution pattern wherein changes in the number of chromosomes and the distribution of AT and GC base pairs were recorded. The cells studied in the plantlets of the first regeneration (23.33 %) showed deviant number of chromosome which was increased to 33.33 % and 40 % in the plantlets of the second and the third regenerations, respectively. Also, 4',6-diamidino-2-phenylindole (DAPI)(+) and chromomycin A3 (CMA)(+) binding sites, on an average of 5.74 ± 0.47 and 5.00 ± 0.30, were observed in the plantlets of the first regeneration. Subsequently, DAPI(+) binding sites were increased to 6.61 ± 0.39 and 6.74 ± 0.57 in the plantlets of the second and the third regenerations, respectively, with a corresponding decrease in the CMA(+) binding sites (4.63 ± 0.45 and 4.16 ± 0.47 CMA(+) sites in the plantlets of the second and the third regenerations, respectively). The study reveals an increase in cytological variations in the morphologically similar micropropagated plants of N. khasiana with the subsequent regenerations which further necessitate the determination of genetic integrity of micropropagated plants.

Micropropagation of Eucalyptus camaldulensis for the production of rejuvenated stock plants for microcuttings propagation and genetic fidelity assessment

Adventitious rooting, a key step in clonal propagation is affected by tree maturation. Micropropagation followed by microcuttings propagation has the potential to rejuvenate the clones thereby enhancing the rooting potential and minimize intra clonal variation. In this study, 33 superior performing Eucalyptus camaldulensis clones were propagated by rooting of stem cuttings (SCs) and micropropagation. Micropropagated plantlets were used as stock plants for microcutting propagation. Rooting of SCs and micropropagation was carried out with the coppice shoot cuttings and axillary buds respectively, obtained from approximately fourteen year old trees that had undergone one vegetative propagation cycle. The adventitious rooting recorded was significantly higher in micropropagation (24.8–100 %) and microcuttings (43–95 %) than SCs method (9.3–75.5 %). Studies on ontogeny of adventitious rooting showed the emergence of root primordium from the phloem region and root initials were noticed within 5–9 days after auxin treatment. Further, molecular marker analysis showed genetic uniformity except for two ramets, detected using inter simple sequence repeats (ISSR) markers and suitable corrective measures were taken to avoid entry of such plantlets for mass multiplication. This study demonstrates the importance of integration of micropropagation and microcuttings production for rejuvenation and mass multiplication. Although current rejuvenation and root induction treatments favored adventitious rooting, the basic mechanisms involved in rejuvenation and adventitious rooting need to be explored for hassle free industrial rooting process, consequently cost effective propagation.

Assessment of factors on shoot proliferation potential of nodal explants of Phyllanthus fraternus and assessment of genetic fidelity of micropropagated plants using RAPD marker

Phyllanthus fraternus is an important medicinal plant, popularly known for its hepatoprotective and antiviral activities since ancient times. Various physiological factors like carbon sources, concentration of agar, pH of the media and effect of season of explants collection were optimized for high frequency regeneration of P. fraternus. The frequency of regeneration, average number and length of shoots were highly influenced by the type and concentration of carbon sources (monosaccharides and disaccharides, 1 to 4%), agar concentration (0.2 to 1%) and pH (4.5 to 6.8) of the media. Media containing 3% sucrose, 0.6% agar at pH 5.8 was best for regeneration. Seasonal variation of explants collection significantly affected the axillary shoots proliferation from explants and best proliferation was observed from explants collected during April to June. Genetic fidelity of regenerated plants was assessed by random amplified polymorphic DNA markers. No polymorphism was detected in micropropagated plants and the mother plant, revealing the genetic homogeneity of the in vitro raised plantlets. This is the first report regarding establishment of genetic fidelity of micropropagated P. fraternus plants, which could be successfully applied for the mass multiplication, germplasm conservation and further genetic transformation assays to meet the ever increasing demand of this medicinally potent plant for industrial and pharmaceutical uses.

High efficiency cyclic production of secondary somatic embryos and ISSR based assessment of genetic fidelity among the emblings in Calliandra tweedii (Benth.)

A high frequency secondary somatic embryogenesis and plant regeneration have been achieved in Calliandra tweedii (Benth.) through internodal segments excised from 5-year-old shrub on Murashige and Skoog (MS) medium enriched with different concentrations of 2-isopentenyl adenine (2iP). Somatic embryos were differentiated directly from cut ends, epidermal crevices as well as indirectly through callus. Such primary embryos when subcultured on MS medium augmented with 2-isopentenyl adenine (2iP), N6 benzyladenine (BA), 2,4-dichlorophenoxy acetic acid (2,4-D), α-naphthaleneacetic acid (NAA) in different concentrations (0.1, 0.5, 1, 1.5, 5, 10, and 20μM) individually, redifferentiated numerous secondary somatic embryos of different stages, i.e., globular, heart-shaped, torpedo and cotyledonary types within 6 weeks of culture. The somatic embryos originated either from radicular or plumular parts or from all over the surfaces. Of the aforesaid growth regulators, 1μM 2iP proved to be the best for recurrent production of an average of 19.44±0.52 cotyledonary somatic embryos per culture within 6 weeks. The embryogenic clumps (100mg per culture tube) comprising the pre cotyledonary stages were transferred to different concentrations (1, 2.5, 5, 7, 10, 20, and 50μM) of abscisic acid (ABA) for their maturation and preventing precocious germination. ABA at 5μM proved optimum for developing an average of 15.87±0.31 normal cotyledonary embryos per embryogenic clumps within 4 weeks. These bipolar mature embryos after transfer to half-strength MS medium germinated into plantlets. The genetic uniformity of the emblings was established employing 14 inter simple sequence repeat (ISSR) marker where 96.1% of the bands generated were monomorphic and similar with those of stock plant with only 2% variation among the plantlets.

Micropropagtion of Terminalia bellerica from nodal explants of mature tree and assessment of genetic fidelity using ISSR and RAPD markers.

The present study reports an efficient in vitro micropropagation protocol for a medicinally important tree, Terminalia bellerica Roxb. from nodal segments of a 30years old tree. Nodal segments taken from the mature tree in March-April and cultured on half strength MS medium gave the best shoot bud proliferation response. Combinations of serial transfer technique (ST) and incorporation of antioxidants (AO) [polyvinylpyrrolidone, PVP (50mgl(-1)) + ascorbic acid (100mgl(-1)) + citric acid (10mgl(-1))] in the culture medium aided to minimize browning and improve explant survival during shoot bud induction. Highest multiplication of shoots was achieved on medium supplemented with 6-benzyladenine (BA, 8.8μM) and α-naphthalene acetic acid (NAA, 2.6μM) in addition to antioxidants. Shoot elongation was obtained on MS medium containing BA (4.4μM) + phloroglucinol (PG, 3.9μM). Elongated shoots were transferred to half strength MS medium containing indole-3-butyric acid (IBA, 2.5μM) for root development. The acclimatization of plantlets was carried out under greenhouse conditions. The genetic fidelity of the regenerated plants was checked using inter simple sequence repeats (ISSR) and randomly amplified polymorphic DNA (RAPD) analysis. Comparison of the bands among the regenerants and mother plant confirmed true-to-type clonal plants.

Improved cryopreservation of oil palm (Elaeis guineensis Jacq.) polyembryoids using droplet vitrification approach and assessment of genetic fidelity.

In the present study, polyembryoids of oil palm (Elaeis guineensis Jacq.) were cryopreserved with successful revival of 68 % for the first time using the droplet vitrification technique. Excised polyembryoids (3-5-mm diameter) from 3-month-old in vitro cultures were pre-cultured for 12 h in liquid Murashige and Skoog medium supplemented with 0.5 M sucrose. The polyembryoids were osmoprotected in loading solution [10% (w/v) dimethyl sulphoxide (DMSO) plus 0.7 M sucrose] for 30 min at room temperature and then placed on aluminium strips where they were individually drenched in chilled droplets of vitrification solution (PVS2) [30% (w/v) glycerol plus 15% (w/v) ethylene glycol (EG) plus 15% (w/v) DMSO plus 0.4 M sucrose] for 10 min. The aluminium strips were enclosed in cryovials which were then plunged quickly into liquid nitrogen and kept there for 1 h. The polyembryoids were then thawed and unloaded (using 1.2 M sucrose solution) with subsequent transfer to regeneration medium and stored in zero irradiance. Following for 10 days of storage, polyembryoids were cultured under 16 h photoperiod of 50 μmol m(-2) s(-1) photosynthetic photon flux density, at 23 ± 1 °C. Post-thaw growth recovery of 68% was recorded within 2 weeks of culture, and new shoot development was observed at 4 weeks of growth. Scanning electron microscopy revealed that successful regeneration of cryopreserved polyembryoids was related to maintenance of cellular integrity, presumably through PVS2 exposure for 10 min. The present study demonstrated that cryopreservation by droplet vitrification enhanced the regeneration percentages of oil palm in comparison with the conventional vitrification method previously reported.

Development of an efficient protocol for plant regeneration from nodal explants of recalcitrant bamboo (Bambusa arundinacea Retz. Willd) and assessment of genetic fidelity by DNA markers

The present study describes an efficient method for in vitro plant regeneration in B. arundinacea through axillary shoot bud proliferation. Nodal explants were excised, cultured on MS medium containing different concentrations of 6-benzylaminopurine (BAP), kinetin (KIN) (0.5–5.0 mg l−1) alone and/or in combinations with KIN/BAP (0.5 mg l−1). The highest frequency (91.5 %) of multiple shoot bud induction with maximum number of shoots (85 shoots/explant) was noticed on MS medium + 3.0 mg l−1 BAP + 0.5 mg l−1 KIN. The regenerated multiple shoots were elongated on MS medium + 4.0 mg l−1 KIN + 2.0 mg l−1 gibberellic acid (GA3) with maximum shoot length (4.9 cm). The elongated shoots were transferred to MS medium containing indole-3 butyric acid (IBA; 0.5–5.0 mg l−1) alone and/or in combination with 0.5 mg l−1 KIN and BAP. Highest frequency of rooting (75 %) was obtained on half-strength MS medium + 2.0 mg l−1 IBA + 0.5 mg l−1 KIN. After hardening, the plantlets were shifted to the green house and subsequently established in the field conditions with 90 % survival rate. random amplified polymorphic DNA (RAPD) markers were used to evaluate the genetic stability of the regenerants. RAPD profiles generated from the regenerated plants were found to be monomorphic, similar to the control. Results confirmed that the regenerated plants were true-to-type in nature and the developed micropropagation protocol could be used for large scale plant production of B. arundinacea.

In Vitro Propagation, Encapsulation, and Genetic Fidelity Analysis of Terminalia arjuna: a Cardioprotective Medicinal Tree

The present study described an improved and reproducible in vitro regeneration system for Terminalia arjuna using nodal segment explants obtained from a mature plant. Shoot tips excised from in vitro proliferated shoots were encapsulated in 3% sodium alginate and 100mM CaCl2[Symbol: see text]2H2O for the development of synthetic seeds which may be applicable in short-term storage and germplasm exchange of elite genotype. Shoot multiplication was significantly influenced by a number of factors, namely types and concentrations of plant growth regulators, medium composition, repeated transfer of mother explants, subculturing of in vitro regenerated shoot clumps, agar concentrations, and temperature. Maximum numbers of shoots (16.50 ± 3.67) were observed on modified Murashige and Skoog (MMS) medium containing 0.5mgl(-1) of benzylaminopurine (BAP) and 0.1mgl(-1) of naphthalene acetic acid (NAA). To shortening the regeneration pathway, rooting of micropropagated shoots under in vitro condition was excluded and an experiment on ex vitro rooting was conducted and it was observed that the highest percentage of shoots rooted ex vitro when treated with indole-3-butyric acid (IBA, 250mgl(-1)) + 2-naphthoxy acetic acid (NOA, 250mgl(-1)) for 5min. The well-developed ex vitro rooted shoots were acclimatized successfully in soilrite under greenhouse conditions with 80% survival of plants. Randomly amplified polymorphic DNA (RAPD) analysis confirmed that all the regenerated plants were genetically identical to the mother plant, suggesting the absence of detectable genetic variation in the regenerated plantlets. To the best of our knowledge, this is the first report on synthetic seed production as well as ex vitro rooting and genetic fidelity assessment of micropropagated shoots of T. arjuna.

Plant regeneration from leaf explants of Aloe barbadensis Mill. and genetic fidelity assessment through DNA markers.

An efficient plant regeneration protocol was developed from leaf explants of Aloe barbadensis Mill on Murashige and Skoog's (MS) medium supplemented with 2.0mg/l 6-benzyladenine (BA) or Kinetin (Kn), 0.25-0.5mg/l NAA (1-napthalene acetic acid) and 3% (w/v) sucrose within 4weeks of culture. The maximum number of shoot buds were obtained on MS medium supplemented with 2.0mg/l BA, 0.5mg/l NAA, 40mg/l Ads (adenine sulphate) within 4-6weeks of subculture. Inclusion of 0.25-0.50mg/l gibberellic acid into the medium, the shoot buds became elongated. Repeated subculture on regeneration medium induces higher rate of shoot regeneration. The root induction from excised microshoots was achieved on half-strength MS medium supplemented with 0.25-1.0mg/l NAA or indole-3-butyric acid (IBA) and 2% (w/v) sucrose. Maximum percentage of rooting was achieved on medium having 0.5mg/l NAA with 3% (w/v) sucrose. About 80% of in vitro raised plantlets were hardened in the greenhouse and successfully established in the soil. Both Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeat (ISSR) markers were used to detect the variability among the regenerated plants developed in vitro. The results showed that there was no polymorphism among the regenerated plantlets. This study will help for propagation of quality planting material of Aloe barbadensis for commercialization.

Somatic embryogenesis from mature zygotic embryos of Distylium chinense (Fr.) Diels and assessment of genetic fidelity of regenerated plants by SRAP markers

The objective was to establish an efficient regeneration protocol for Distylium chinense based on somatic embryogenesis and evaluate the genetic stability of plants regenerated in vitro. To induce callus mature zygotic embryos were cultured on Murashige and Skoog’s (MS) medium that was supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and N6-benzyladenine (BA). After 20 days, the highest rate of callus formation (88.9 %) occurred on MS medium supplemented with 0.5 mg l−1 2,4-D and 0.1 mg l−1 BA. It was observed that light-yellow, compact, dry, nodular embryogenic calli had formed. These calli were then subcultured on fresh MS medium supplemented with 0.1 mg l−1 BA and 0.5 mg l−1 α-naphthaleneacetic acid (NAA) for proliferation for an additional 30 days. To induce somatic embryos and plant regeneration, the embryogenic callus was transferred to fresh MS medium that was supplemented with different concentrations of BA and NAA. After 30 days, 0.5 mg l−1 BA in combination with 0.5 mg l−1 NAA produced the best result in terms of somatic embryogenesis (%), shoot differentiation (%), number of shoots per callus and shoot length. Next, the plantlets were transferred to the field for 5 weeks and a 95 % survival rate was observed. The sequence-related amplified polymorphism markers confirmed genetic stability of plants regenerated in vitro. To our knowledge, this is the first report that describes a plant regeneration protocol for D. chinense via somatic embryogenesis to be used for germplasm conservation and commercial cultivation.