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In the present study, a simple, eco-friendly, biogenic protocol for synthesis of silver nanoparticles (AgNPs) using leaf extract of in vitro banana plantlets (Musa acuminata Colla cv. Grand Nain) was established. The synthesized AgNPs were characterized by X-ray diffraction, transmission electron microscopy, scanning electron microscopy, and fourier transform-infrared spectroscopy. Shoot tips of stevia (Stevia rebaudiana Bert.) were cultured on three forms of regeneration medium (Murashige and Skoog basal medium + 0.5 mg. L−1 6-benzyladenine) i.e. 'semi-solid', 'liquid', and 'semi-solid followed by liquid' supplemented with 100–400 µM of AgNPs to study the effect of different concentrations of AgNPs along with different forms of medium on plant growth and rebaudioside-A content in stevia under in vitro conditions. Among different concentrations, 400 µM AgNP showed the best growth response irrespective of different forms of medium. Nonetheless, the overall best growth response was recorded in 'semi-solid followed by liquid' medium. High performance liquid chromatography analysis of regenerants showed gradual incline in rebaudioside-A content with the increase in AgNP concentrations. Regenerants from medium containing 400 µM AgNPs produced the highest rebaudioside-A of 334.8 mg. g−1 dry weight (>2-fold) while control ones produced 154.3 mg. g−1 dry weight. Genetic fidelity of AgNP-treated and control regenerants was confirmed using 20 inter simple sequence repeats and start codon targeted primers. The present study can be useful for commercial-scale propagation of stevia ensuring higher fresh and dry biomass as well as ameliorated rebaudioside-A production. • First report on green synthesis and characterization of silver nanoparticles (AgNPs) from in vitro grown banana leaf. • Among three forms, 'semi-solid followed by liquid' medium showed superior growth of stevia. • Ameliorated biomass and rebaudioside-A production with rising AgNP concentrations in stevia. • AgNP-treated regenerants via ISSR and SCoT markers confirmed 100% genetic fidelity. [ABSTRACT FROM AUTHOR]

Orthosiphon stamineus has been widely used as traditional remedy for various illnesses and diseases, such as cardiovascular diseases and epileptic seizures. In this study, direct regeneration through nodal segment of this species was attempted using Kinetin (6-Furfurylaminopurine) and IAA (indole-3-acetic acid). Optimum regeneration media was identified as MS media supplemented with 2.0 mg L−1 Kin plus 0.5 mg L−1 IAA. This yielded the highest number of shoots (5.57 ± 0.42) and leaves (20.53 ± 1.91) per explant. Acclimatization of the resulting in vitro regenerants was successful in all potting mixtures tested. However, potting mixture PF (1:1:1 ratio of black soil/red soil/compost) was identified as the best medium for acclimatization of this species, as it yielded 100% survival percentage after 90 days of acclimatization. Ten in vitro regenerants of O. stamineus were randomly collected after the third subculture and subjected to genetic variation analysis using inter-simple sequence repeat (ISSR) markers. Out of 20 ISSR markers tested, 10 working primers were observed to produce satisfactory amplification of bands, with an average of 7.11 bands per primer. A total of 610 bands were produced by the 10 primers. The percentage of polymorphism was observed to be very low, yielding only 7.32% polymorphism among all samples. Jaccard dissimilarity analysis was also conducted and very low genetic distance (about 0.1) was found among the in vitro regenerants and between the regenerants with the mother plant, thus ascertaining the clonal nature of the plantlets produced in this study.

An efficient and reproducible protocol for in vitro plant regeneration was developed for Lawsonia inermis L. using cotyledonary node explant derived from axenic seedlings. Highest shoot proliferation frequency (ca 96.6%) was achieved on Murashige and Skoog's, 1962 (MS) basal medium supplemented with 8.88 μM 6-Benzyladenine (BA) + 2.68 μM Napthalene acetic acid (NAA). Up-scaling of shoots was carried out using in vitro nodes on MS medium supplemented with 4.44 μM BA. So overall, an average of 238 shoots was produced at 75 days. Of the four different forms of cotyledonary node explants evaluated, highest shoot multiplication was observed in cotyledonary node explant with two whole cotyledons. In vitro regenerated shoots were best rooted (ca 34.3 roots / shoot) on ½ MS medium devoid of any growth regulator. The plantlets were successfully acclimated in sand:soil:: 1:1and established in the garden soil. Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) analysis revealed a homogeneous amplification profile for all micropropagated plants validating the genetic fidelity of the in vitro-regenerated plants and supporting the regeneration protocol for economic commercial exploitation. [ABSTRACT FROM AUTHOR]

An efficient mass propagation system for a rapid delivery of pro-vitamin A cassava varieties to farmers is essential for acquiring food sufficiency and reducing vitamin A deficiency in Africa. The study investigated multiple shoot inductions from axillary buds of three pro-vitamin A enriched cassava varieties using a combination of 10 mg/l 6-benzylaminopurine (BAP) and 0.1-0.5 mg/l naphthalene acetic acid (NAA). Growth, photosynthetic pigment content and molecular stability of the regenerated plants with random amplified polymorphic DNA (RAPD) markers are also assessed. Shootbud formation in TMS 30572 variety was found to be 8.9% higher than in the three pro-vitamin A varieties. But, the number of regenerated shoots was higher by 7.3% in pro-vitamin A varieties than in TMS 30572. The formation of roots was outstanding in UMUCASS 38. Root carotene contents of regenerated and mother plants were not different. The highest number of shootbuds per explant and frequency of regenerated shoots were obtained through a medium containing 10 mg/l BAP and 0.3 mg/l NAA. The RAPD analysis of regenerated plants showed a uniform profile of 1628 bands among the regenerants and mother plants. The study concluded that a combination of 10 mg/l BAP and 0.3 mg/l NAA enhances a multiple shoot induction with no genetic infidelity of regenerated plants. [ABSTRACT FROM AUTHOR]

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In vitro clonal propagation of small fruit species has been one of the most broadly exploited approaches in biotechnology. However, the most critical aspect of this propagation technology has been the maintenance of genetic fidelity of tissue culture-raised plants. In the present study, three techniques were deployed in the assessment of genetic fidelity of micropropagated plants (TC plants) of blackberry cultivar ‘Čačanska Bestrna’ and raspberry cultivar ‘Meeker’ in the second year after planting in the open field, in comparison to plants originating from the standard planting material (ST plants). Flow cytometry analysis deployed to estimate the DNA ploidy level and relative nuclear DNA content in TC and ST plants revealed no significant differences in nuclear DNA content between plants of different origin. Chromosome counting in root/shoot tip meristems also showed a normal tetraploid chromosome number (2 n = 4 x = 28) in TC blackberry plants and a normal diploid chromosome number (2 n = 2 x = 14) in TC raspberry plants. Polyacrylamide gel electrophoresis of peroxidase (POX) extracted from the leaf tissue revealed no differences in POX profiles between TC and ST plants either. The results obtained in this study verify the suitability of micropropagation by axillary branching for commercial exploitation in blackberry and raspberry. [ABSTRACT FROM AUTHOR]

Key message : We induced somatic embryogenesis and regenerated plants from the somatic tissues of a 700-year-old Kalopanax septemlobus tree. Embryonic calli were induced from juvenile tissues of grafted plants by pre-treating them with 1 M sucrose solution. Many normal plants were produced. Abstract: Asexual propagation via somatic embryogenesis supports tree improvement programs. However, the induction of somatic embryogenesis from mature trees is still recalcitrant in many woody species with the exception of a few conifer and oak species. Previously, we developed a somatic embryogenesis protocol for a mature (40 years) Kalopanax septemlobus tree using rejuvenated tissues obtained by serial grafting. In the present study, we performed somatic embryogenesis and plant regeneration using a 700-year-old K. septemlobus tree. Calli were induced from young leaf and petiole explants of grafted plants by pre-treating them with 1 M sucrose solution. The induction rate of the embryogenic calli (EC) was less than 0.5%. However, the EC proliferated well and readily formed somatic embryos (SE), which were then converted to plantlets using routine protocols. The somatic EC did not differ from EC obtained from zygotic embryos with respect to SE formation, germination, and plant conversion. The genetic identities of regenerated plantlets were verified by comparison with the mother tree by chromosome counting, flow cytometry, and inter simple sequence repeat marker analysis. [ABSTRACT FROM AUTHOR]

The aim of this study was to develop an efficient protocol for the in vitro regeneration of chayote ( Sechium edule ), an important Mexican crop and the evaluation of the genetic fidelity. This species produces recalcitrant seeds, which complicates germplasm storage and makes in vitro conservation a desirable alternative. Proliferation of axillary shoots was induced from axenic nodal segments obtained from in vitro germinated seedlings. Nodal segments were cultured on MS medium supplemented with 0.1 mg/L 6-benzylaminopurine (BA), producing 8.0 ± 0.4 shoots per explant with a regeneration frequency of 92%. Plant regeneration by organogenesis was optimally achieved when leaf and petiole explants were cultured on MS medium supplemented with 0.1 mg/L BA and 0.05 mg/L gibberellic acid (GA3), obtaining an average of 5.3 ± 1.9 shoots per explant. All of the regenerated shoots originated directly from petioles without previous formation of callus. Adventitious shoots were successfully rooted in MS medium without growth regulators after 2 weeks. Since somaclonal variation is a potential hindrance to the in vitro propagation of plants, genetic stability was evaluated through RAMP markers using 9 combinations of RAPD and ISSR primers. The amplification products obtained from the regenerated plants were electrophoresed and showed banding patterns similar to that of the mother plant, demonstrating the homogeneity of the individuals obtained through the regeneration protocols reported here. [ABSTRACT FROM AUTHOR]

In the present study, we develop an efficient and reproducible in vitro regeneration system for two cultivars viz. , Jamila and Tomaland of Solanum lycopersicum L., an economically important vegetable crop throughout the world. Sterilization of seeds with 2.5% (v/v) NaOCl was found to be most effective, about 97% of seeds germinated on cotton in magenta box moistened with sterile half strength (½)Murashige and Skoog (MS) medium. Regeneration efficiency of cotyledonary leaf (CL) and cotyledonary node (CN) explants derived from 08 days old aseptic seedling were assessed on MS medium supplemented with different concentrations of auxins and cytokinin. CL explants were found more responsive in comparison to CN in both the cultivars. Types of basal media were also assessed and found to have a significant effect on shoot regeneration. Highest regeneration frequency and maximum number of shoots were standardized from CL explants on MS medium supplied with 6-benzyl adenine (BA; 5.0 µM), indole-3-butyric acid (IBA; 2.5 µM) and Kinetin (Kin; 10.0 µM). In vitro regenerated microshoots were rooted on ½MS medium containing 0.5 µM indole-3-butyric acid (IBA). Regenerated plantlets with well-developed roots and shoot system were successfully acclimated to ex vitro condition. Genetic uniformity of tissue culture raised plantlets was first time evaluated using flow cytometry and single primer amplification reaction (SPAR) methods viz ., DAMD and ISSR. No significant changes in ploidy level and nuclear DNA content profile were observed between in vitro propagated plants and normal plants of both the cultivars. Similarly, the SPAR analysis also revealed monomorphic banding patterns in regenerated plantlets of S. lycopersicum verifying their genetic uniformity and clonal fidelity. This efficient regeneration system can be used as a fast and reproducible method for genetic transformation of this important vegetable crop. [ABSTRACT FROM AUTHOR]

A simple and efficient regeneration protocol was developed for watermelon from cotyledonary node explants excised from 7-day-old in vitro grown seedlings. This study describes the effect of amino acids and polyamines (PAs) along with plant growth regulators (PGRs) on multiple shoot induction and rooting. The highest number of multiple shoots (46.43 shoots/explant) was obtained from cotyledonary node and they were also elongated (6.3 cm/shoot) on MS medium supplemented with 1 mg l N -Benzyladenine (BA), 5 mg l leucine, and 10 mg l spermidine. The elongated shoots developed profuse roots (23.03 roots/shoot) in MS medium containing 1 mg l indole-3-butyric acid (IBA), 5 mg l isoleucine, and 10 mg l putrescine. All the rooted plantlets were successfully hardened and acclimatized in the greenhouse with a survival rate of 98%. The present study described an efficient method to obtain a 1.5-fold increase in the number of shoots, compared with the available regeneration protocols for watermelon. The plants developed in this study showed fivefold higher photosynthetic pigments compared to the control plants. The genetic fidelity of the regenerated plants was evaluated by SCoT and RAPD marker analyses, and banding patterns confirmed the true-to-type nature of in vitro regenerated plants. [ABSTRACT FROM AUTHOR]

Present study reports successful in vitro clonal propagation of a potential medicinal plant, Cassia alata using mature nodal explants. Murashige and Skoog (MS) medium supplemented with different concentrations (0.5, 2.5, 5.0, 7.5, 10.0 and 12.5 μM) of 6-benzyladenine (BA), kinetin and 2-isopentenyl adenine (2-iP) singly as well as in combination with different auxins, α-naphthalene acetic acid (NAA), Indole-3-butyric acid (IBA) or Indole-3-acetic acid (IAA) (0.1, 0.5 and 1.0 μM) were used. MS medium enriched with 7.5 μM BA and 0.5 μM NAA yielded the highest regeneration frequency (92 %) with maximum multiple shoots (12.3 ± 0.6) and shoot length (4.7 ± 0.1 cm) after 12 weeks of culture. Shoots were rooted best on full MS containing 0.5 μM IBA. Ex vitro rooting of in vitro derived microshoots was also achieved in 150 μM IBA treatment for 20 min followed by transfer to thermocol cups containing sterile Soilrite™. About 85 % plantlets survived acclimatization procedure to the field. The genetic fidelity of in vitro regenerated plants was analyzed using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. Of the 20 RAPD primers, 18 primers produced clear, reproducible and scorable bands while out of 13 ISSR primers screened, only ten generated well-defined and scorable bands in all the tested plants. A total of 69 and 71 bands were scored with an average of 3.8 and 7.1 bands per primer for RAPD and ISSR primers respectively. All banding profiles from micropropagated plants were monomorphic and similar to of the mother plant, thus confirming the true-to-type nature of the in vitro-raised clones. [ABSTRACT FROM AUTHOR]

Key message: This paper provides authentic information on the micropropagation and genetic fidelity assessment methods, which can be used for conservation and quality assessment of endangered medicinal plant Zanthoxylum armatum. Abstract: An in vitro propagation protocol for Zanthoxylum armatum (local name-Timur, Family-Rutaceae), an endangered medicinal plant of the Himalayan region, has been developed through culture of nodal explants. Use of Murashige and Skoog (MS) medium, supplemented with 12.0 µM 6-benzylaminopurine (BAP), 0.5 µM indole-3-acetic acid (IAA), and 0.5 µM gibberellic acid (GA), resulted in good shoot proliferation (4.78 ± 0.18 shoots/explant) and shoot length (3.46 ± 0.05 cm). One hundred percent rooting was achieved through pulse treatment of shoots on full-strength MS medium supplemented with 50.0 µM indole-3-butyric acid (IBA) for 12 h, followed by subsequent transfer to half strength MS medium without plant growth regulators (PGRs). The plant survival was found to be 75% following acclimation. Plants started to flower after 1 year of transfer to soil under nursery conditions. Genetic stability of the regenerated plants was assessed using random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) markers. Results confirmed monomorphic nature of regenerated plants, matching with that of the mother plant, and thus genetic fidelity of the micropropagated plants. This is the first report for micropropagation of Z. armatum. Following further field trials, the protocol can be used for raising quality planting material for commercial cultivation which should also help in its conservation. [ABSTRACT FROM AUTHOR]

The aim of this study was to produce sugarcane plantlets from cell suspension culture and study its genetic fidelity using molecular markers. The study was carried out using sugarcane varieties Co 86032 and Q117. Callus cultures of both the varieties were optimized using six different callus induction media. After screening the growth response of callus on six different callus inductionmedia, itwas observed that medium no. VI supplemented with 500 mg l-1 of each PVP, Casein hydrolysate and MES buffer showed high amounts of callus in Co 86032 (79.66 ± 0.44%) and Q117 (82.83 ± 1.69%). Addition of PEG 8000 at 2.5% to this medium had a profound impact on inducing somatic embryogenesis in Co 86032 (54.66 ± 1.76%) and Q117 (66.66 ± 2.60%) as compare to control (24.33 ± 1.76%) and (27.33 ± 2.73%), respectively. Cell suspension cultures were established by culturing embryogenic calli in liquid medium showed well established suspension cultures with fever cell aggregates. There was negligible cell division during initial 2 days of incubation and cell count increased rapidly between 2 and 8 days. Further incubation beyond 8 days resulted in a decrease in cell viability. Enhanced callus proliferation in Q117 while enhanced shoot regeneration in Co 86032 was observed from cell suspension culture. The clonal fidelity of in vitro regenerated plants was assessed by using RAPD and ISSR markers. Analysis of the ten RAPD markers indicated that 90.48 and 86.95%true-to-type regenerated plantlets in Co 86032 and Q117, respectively. However, in the ISSRmarkers, Co 86032 did not show any polymorphism and in the Q117, 92.18% true-to-type plantlets were found. These results confirmed that somaclonal variation occurs during the process of indirect organogenesis and RAPD and ISSR marker based molecular analysis is a suitable method for an early detection of variation in sugarcane. [ABSTRACT FROM AUTHOR]

In the present study we have developed an efficient and effective method of synthetic seed production and its two-way germination system ofVitex trifolia, for easy transport of the propagules and efficient utilization of itsin vitroregeneration system. Nodal segments harvested from 8-week-oldin vitrocultures were encapsulated in calcium alginate beads. Three percent (w/v) Na-alginate polymerized in 100 mmol/L CaCl2.2H2O for 30 min produced clear and uniform beads. Germination of encapsulated beads with shoot and roots was achieved on Murashige and Skoog (MS) medium augmented with 6-furfurylaminopurine (KN, 2.5 µmol/L) + α-naphthalene acetic acid (NAA, 1.0 µmol/L). For multiple shoot production, synseeds were incubated on 6-benzyladenine (BA, 5.0 µmol/L) + NAA (0.5 µmol/L) augmented MS medium followed byin vitrorooting on MS + indole-3-butyric acid (IBA, 1.0 µmol/L). The synseeds produced retained about 90% regeneration potential even after 4 weeks of storage at 4°C. Genetic stability of the regenerated plants was evaluated using 13 inter simple sequence repeats (ISSR) primers. The study thus provides an efficient system for production of synthetic seeds, their storage and subsequent conversion into genetically identical plants. [ABSTRACT FROM PUBLISHER]

Date palm is propagated by offshoot and tissue culture to produce plants that are true- to- type, which leads to decreased genetic diversity. Cryopreservation is used for the preservation of genetic resources of a wide range of plant species for short- to long-term storage. Callus induction was achieved for four cultivars (Ajwa, Khodary, Ruthana, and Sukary) on MS medium with 2,4-dichlorophenoxyacetic acid (2,4-D) (2.5 mg/l) and α-naphthaleneacetic acid (NAA) (5-7.5 mg/l) in combination with benzyladenine (BA) (0.5-1 mg/l). The calli were cryopreserved by encapsulation--vitrification and dehydration methods. Survival and regeneration after cryopreservation in liquid nitrogen ranged from 51--57% and 29.66--41.33%, respectively, among the cultivars with encapsulation--vitrification method. However, survival and regeneration was much low with dehydration method as compared to encapsulation--vitrification and it was ranged from 16-24.66% and 27-33.66%, respectively. Genetic fidelity was assessed using SCoT markers in regenerated plantlets after cryopreservation and compared to mother plants as well as with tissue culture raised plantlets. Out of a total of 118 amplicons produced by 18 SCoT primers, 114 were monomorphic and the remaining were polymorphic. This low genetic variation indicates the clonal genetic stability of the regrown plantlets after cryopreservation. [ABSTRACT FROM AUTHOR]

A simple method has been developed for clonal propagation of mature trees of Tecomella undulata (Sm.) Seem, a medicinally important deciduous timber tree of hot arid regions, via multiple shoot proliferation from axillary buds after examining the role of season influences and physico-chemical conditions on micropropagation. Spring season (March-April) was the best period for contamination free establishment of explants and maximum sprouting of healthy axillary buds. Shoots proliferated directly from the explant nodes cultured on Murashige and Skoog's medium containing cytokinins, BAP supporting better growth compared to kinetin during shoot induction as well as multiplication phase. Cytokinin concentration influenced the bud induction frequency and optimal response of 2.6 buds per explant was achieved in 86.66% explants on media supplemented with 10 µM BAP. Stunted shoot buds with excessive callus were observed when cytokinin concentration was increased beyond optimal levels. Ascorbic acid (50 mg/l), arginine and citric acid (25 mg/l each) were added to proliferation and multiplication media for reducing callus proliferation and better shoot growth. Among the media (B5, MS, NN, WPM and SH) tested, SH was best for shoot multiplication. Shoot cultures were multiplied by regular subculture of axillary shoots on SH medium containing 5.0 µM each of BAP and kinetin. Shoots produced roots when cultured on ½× SH medium + 10 μM IBA. Regenerated plantlets were successfully transferred to field after hardening and acclimatization. Genetic homogeneity of tissue culture raised plants was confirmed by generation of monomorphic DNA fragments with Start codon targeted and intersimple sequence repeat (ISSR) markers. [ABSTRACT FROM AUTHOR]

Protoplast isolation and subsequent plant regeneration of Albizia julibrissin was achieved from leaf and callus explants. Leaf tissue from 4 to 5-week-old in vitro seedlings was the best source for high-yield protoplast isolation. This approach produced 7.77 × 10 protoplasts (Pp) per gram fresh weight with 94 % viability; after 60 min pre-plasmolysis with 0.7 M sorbitol followed by digestion in a solution of cell and protoplast wash plus 0.7 M mannitol, 1.5 % cellulase Onozuka R10, and 1 % pectolyase Y-23 for 6 h. Liquid Kao and Michayluk medium containing 2.7 μM α-naphthaleneacetic acid (NAA) and 2.2 μM 6-benzylaminopurine (BA) was best for sustained cell division and microcolony formation from both leaf- and callus-derived protoplasts at a density of 3-5 × 10 Pp ml. Protoplast-derived microcalli became visible after 3-4 weeks on semi-solid medium of the same composition. Microcalli were then cultured on Murashige and Skoog (MS) medium containing Gamborg B5 vitamins or woody plant medium supplemented with different concentrations of NAA plus 4.4 μM BA for further growth. Proliferated leaf- and callus-protoplast-derived calli differentiated into microshoots on MS medium containing 13.2 μM BA plus 4.6 μM zeatin after 2-3 weeks, with an overall shoot organogenesis efficiency of 78-93 %. Rooting of microshoots on half-strength MS medium containing 4.9 µM indole-3-butyric acid was successful, and plantlets were acclimatized to the greenhouse with a survival rate of >62 %. Using ten start codon targeted and ten inter-simple sequence repeat primers, the genetic integrity of nine leaf- and six callus-protoplast-based plants was validated along with the mother seedlings. [ABSTRACT FROM AUTHOR]

A reliable protocol has been established for in vitro propagation of Artemisia nilagirica var. nilagirica (Indian wormwood), a valuable medicinal plant from India. A highly proliferating organogenic callus was obtained on Murashige and Skoog (MS) medium supplemented with 2.5 µM IAA when nodal explants were cultured on MS medium supplemented with various growth regulators. Further, highest regeneration frequency (83.3 %) of adventitious shoots was observed, when the callus was sub-cultured on MS medium supplemented with 6-benzylaminopurine (BAP; 2.5 µM) along with 7.5 µM 2-isopentenyl adenine (2-iP). An optimal of 10.16 ± 2.24 shoots were regenerated on medium supplemented with 2.5 µM BAP + 7.5 µM 2-iP. Quarter strength MS medium supplemented with 10 µM IBA was effective for rooting of the shoots. Ex-vitro plants were normal and were established successfully. Cytological and molecular marker studies showed that regenerated plants showed genetic stability in micro-propagated plants. [ABSTRACT FROM AUTHOR]

Stevia rebaudiana Bertoni ( Asteraceae ) contains in its leaves sweeteners such as stevioside and rebaudioside, for which it is commercially valuable. Inefficient sexual and asexual propagation methods do not meet the current demand of propagules required for commercial cultivation. Therefore, in vitro propagation is a promising alternative to solve this issue. However, the protocols currently available involve low numbers of micropropagated shoots and high production costs. Therefore, this study proposes to establish a commercial micropropagation protocol for S. rebaudiana using Recipient for Automated Temporary Immersion (RITA®) aimed at implementing this mass-propagation process at a low cost and in an automated modality. Our findings revealed that the highest number of shoots (18.37) was obtained using 1 mg l − 1 benzyladenine (BA) at an immersion frequency of 2 min every 8 h in 20 ml medium per explant. 100% rooting of shoots was achieved by using MS medium with no plant growth regulators (PGR). 90% of regenerated plantlets were successfully acclimatized under greenhouse conditions. The regenerated plants showed a low percentage of genetic variation (10.4%). This protocol can be applied in the commercial micropropagation of this species. [ABSTRACT FROM AUTHOR]

A viable and simple method for the large scale production of breadfruit (Artocarpus altilis) using tissue culture is used at the Food and Agricultural Research Extension Institute in line with the Food Security policy of Mauritius. Shoot tip buds of Artocarpus altilis are initiated and multiplied on basal micropropagation medium supplemented with 4.4 µM Benzyl adenine (BA). Direct systems of regeneration through the culture of organized meristems normally produce true-to-type plants. However, variations among in vitro raised plants have been widely reported. The aim of this study was to use ISSR markers to confirm the clonal fidelity of in vitro derived plantlets of breadfruit. DNA extraction of Artocarpus altilis was performed using a modified CTAB method and then purified using RNase and phenol treatment. The presence of DNA was verified on agarose gel electrophoresis and its yield and quality were determined by spectrophotometry. An average yield of 307.3 µL DNA was obtained but with some proteins, phenols and polysaccharides contamination was noted. The 63 markers generated by Inter Simple Sequence Repeat (ISSR) markers revealed a mean of 22.2% genetic variation and 77.7% genetic fidelity in the 15 week cultured plantlets and a mean of 1.9% genetic variation and 98.2% genetic fidelity in the 38 weeks cultured plantlets. These findings indicate that shoot meristems can be used as explants for ensuring clonal fidelity of micropropagated A. altilis and confirm the usefulness of ISSR markers as a certification tool for monitoring genetic stability during the regeneration process. [ABSTRACT FROM AUTHOR]

An in vitro protocol has been established for clonal propagation of Nothapodytes nimmoniana which is an important source of Camptothecin (CPT). Elite source was identified based on the chemical potency to accumulate the optimum level of CPT. Different types and concentrations of plant growth regulators were used to study their effect on inducing multiple shoots from the explants regenerated from embryos of N. nimmoniana. Of these, a combination of N6-benzyladenine (0.2 mg L) and Indole-3-butyric acid (IBA) (0.1 mg L) proved optimum for differentiating multiple shoots in 90.6 % of the cultures with an average of 10.24 shoots per explant obtained within 8 weeks of inoculation. Nearly, 92 % of the excised in vitro shoots rooted on half strength Murashige and Skoog (MS) medium containing 0.05 % activated charcoal, supplemented with 1-naphthaleneacetic acid and IBA at 0.1 mg L each. The micropropagated plants were evaluated for their genetic fidelity by employing inter simple sequence repeats (ISSR) markers. Ten individuals, randomly chosen from a population of 145 regenerants, were compared with the donor plant. The regenerated plants were also evaluated for their chemical potency using high-performance liquid chromatography (HPLC) analysis of CPT content. The true-to-type nature of the micropropagated plants was confirmed based on their monomorphic banding profiles with that of the mother plants using ISSR markers. Besides, HPLC evaluation of the CPT content confirmed the existence of chemical uniformity among the regenerated plants and the elite mother plant. [ABSTRACT FROM AUTHOR]

Somatic embryogenic system was developed in Sapindus mukorossi Gaertn. using rachis as explants from a mature tree. Explants showed callus initiation on Murashige and Skoog medium supplemented with TDZ (1-Phenyl-3-(1, 2, 3-thiadiazol-5-yl) urea), zeatin or 6-benzylaminopurine. Induction of somatic embryogenesis was achieved on both MS basal medium and MS medium supplemented with 8.88 µM 6-benzylaminopurine. Hundred percent embryogenesis was observed on MS medium supplemented with 8.88 µM 6-benzylaminopurine with maximum intensity of embryogenesis (51.92 ± 0.40 a). Maximum maturation of somatic embryos (92.86 ± 0.34 a) was observed on induction medium supplemented with 0.0378 µM abscisic and treated for 21 days. Germination of somatic embryos was maximum (77.33 ± 0.58 a) on MS medium supplemented with 8.88 µM 6-benzylaminopurine. In vitro raised plantlets were hardened, acclimatized and transferred to the field. Survival frequency of plantlets was 80 % in field conditions. The genetic fidelity of in vitro regenerated plants was also evaluated and compared with mother plant using random amplified polymorphic DNA and inter simple sequence repeat. Both markers showed similarity in molecular profile of mother plant and in vitro regenerated plants. [ABSTRACT FROM AUTHOR]

Embryogenic cultures have been used in cryopreservation, genetic transformation, propagation, virus elimination, induced mutagenesis and in many other biotechnological applications, providing excellent opportunities for biotechnology advances in grapevine. Unfortunately the efficiency of somatic embryogenesis (SE) is genotype-dependent in addition to showing interaction with explant type used and the plant growth regulator (PGR) composition. In order to identify the interaction of these parameters in SE, we tested eight wine grapevine cultivars, three explant types (ovary, anther/filament and stigma/style) and four PGR combinations in a statistically designed experiment. The genotype was the major determining factor, with embryogenic response varying from 0.1 to 5.1% (about 50-fold difference). For PGR composition of the medium, embryogenesis ranged between 0.5 and 3.3% (a ≈7-fold difference). The explant type was the least important factor with embryogenesis ranging between 0.8% (anther/filament) and 2.3% (ovary)—only a ≈3-fold change. Anther/filament, that had generally been considered to be the most promising explant, surprisingly gave the lowest embryogenesis percentage. Genetic homogeneity of plants developed from in vitro SE was assessed in comparison to mother plants using six inter-simple sequence repeat and ten random amplified polymorphic DNA primers that produced reproducible and clear bands ranging from 150 to 3500 bp. The amplification products were monomorphic across all the regenerated plants and their respective mother plants confirming the genetic homogeneity of the regenerants and demonstrating the suitability of SE for in vitro grapevine germplasm conservation and propagation. [ABSTRACT FROM AUTHOR]

Genetic stability of in vitro plants should be assessed in order to develop proper programs of conservation. Such conservation programs could be developed only after evaluation of genetic variability in the natural populations and the genetic stability or somaclonal variability after conservation. In case of valuable economic plants as fruit trees, vegetables, medicinal plants, the natural variability from populations should be preserved as sources of new cultivars or variations. In order to preserve endangered, vulnerable or endemic plants it is very important to evaluate the genetic variability in natural populations and to preserve this variability as well as individuals or habitats. Genetic variability or stability after plant conservation is usually assessed by DNA-based molecular markers as RAPD, SSR, ISSR, SRAP, RFLP and AFLP. [ABSTRACT FROM AUTHOR]

Low rates of shoot multiplication, clonal instability, poor rooting frequency and high cost of production are major impediments challenging the micropropagation of orchids and other ornamental plants. These problems can be solved with the proper usage of plant growth regulators (PGRs) in the medium. The highest shoot proliferation rate of 21 shoots/explant was recorded in Murashige and Skoog (MS) medium containing 1 mg/l meta-topolin and 0.8 mg/l Putrescine. To date, this is the best frequency of shoot induction for Dendrobium nobile and one of the highest among other orchids. Higher rooting frequency was also recorded with the highest rooting of 10.1 roots/shoot achieved in plants grown in half strength MS media supplemented with 2 mg/l of indole butyric acid and 0.5 mg/l of phloroglucinol. The plantlets were successfully acclimatized in the greenhouse with a survival rate of 82.3% exhibiting normal developmental patterns. The regenerated plantlets were assessed for genetic stability using the molecular markers namely SCoT and IRAP revealing high degree of genetic stability within the micropropagated plants of D. nobile . Being a highly medicinal orchid, the antioxidant potentials of the mother and the micropropagated plants were assessed using DPPH, FRAP and metal chelating assays which revealed a significantly higher antioxidant activity in the micropropagated plants. Rapid multiplication rate, higher genetic stability and significantly higher antioxidant power reported in the present study on D. nobile ensure the use of this micropropagation protocol for ex-situ conservation and commercial exploitation which could also be extended to other important orchid species. [ABSTRACT FROM AUTHOR]

Salvia nemorosa L. is a valuable medicinal and ornamental plant from Lamiaceae. A high-frequency protocol for in vitro regeneration from S. nemorosa leaf explants was developed based on our results. Various plant growth regulators were evaluated for their effects on direct and indirect organogenesis, callogenesis, shoot multiplication, and rooting of explants at different concentrations. The maximum percentage of direct shoot regeneration (97.5%) and the number of shoots per explant (8.6) were obtained on MS medium supplement with 8 µM TDZ plus 8 µM NAA. The result revealed that media amended by 16 µM 2, 4-D with 8 or 16 µM BA was the best treatment for callus induction. Media containing the combination of 8 µM TDZ plus 2 µM NAA provided the highest rate of shoot induction (84%) and the highest number of shoots (5.4) from calli. Regenerated shoots were multiplied efficiently with an average of 7.4 shoots and 32.4 leaves per explant on media containing 12 µM TDZ. In the present study, TDZ and BA treatments led to the highest proliferation rate; however, adenine sulfate was preferred for proliferation because it did not produce hyperhydric plants. The genetic fidelity of 19 plantlets (one donor plant, nine direct and nine indirect regenerants) was appraised by 10 ISSR markers. The molecular analysis revealed that direct organogenesis produced true-to-type plantlets and could be employed for clonal multiplication of Woodland Sage. Indirect regenerants showed polymorphic bands (12.5–33.33%), confirming the somaclonal variation occurrence during indirect organogenesis. Also, HPLC analysis of regenerants showed that two indirect regenerants accumulated higher rosmarinic acid content than mother plants, while three of them accumulated lower rosmarinic acid content. As part of this study, molecular and biochemical markers have been employed to investigate somaclonal variation in in vitro regeneration of the Salvia genus for the first time, which could be utilized to develop reliable methods for multiplying elite lines and achieving high-yield lines. [Display omitted] • A high-frequency protocol for S. nemorosa in vitro regeneration was developed. • TDZ and NAA combination produced plantlets with high genetic and biochemical fidelity. • Adenine Sulphate prevents the Hyperhydricity in regenerated plantlets. • The indirect regeneration affected PAL activity, rosmarinic acid, and genetic content. [ABSTRACT FROM AUTHOR]

Panax ginseng Meyer grows in east Russia and Asia. There is a high demand for this crop due to its medicinal properties. However, its low reproductive efficiency has been a hindrance to the crop's widespread use. This study aims to establish an efficient regeneration and acclimatization system for the crop. The type of basal media and strength were evaluated for their effects on somatic embryogenesis, germination, and regeneration. The highest rate of somatic embryogenesis was achieved for the basal media MS, N6, and GD, with the optimal nitrogen content (≥35 mM) and NH 4 + /NO 3 - ratio (1:2 or 1:4). The full-strength MS medium was the best one for somatic embryo induction. However, the diluted MS medium had a more positive effect on embryo maturation. Additionally, the basal media affected shooting, rooting, and plantlet formation. The germination medium containing 1/2 MS facilitated good shoot development; however, the medium with 1/2 SH yielded outstanding root development. In vitro-grown roots were successfully transferred to soil, and they exhibited a high survival rate (86.3%). Finally, the ISSR marker analysis demonstrated that the regenerated plants were not different from the control. The obtained results provide valuable information for a more efficient micropropagation of various P. ginseng cultivars.

An efficient in vitro protocol for high-frequency polyploidization for the first time in gerbera hybrid (BGC-2019-01) was developed in the present study. Two-week-old in vitro-developed shoots (tips) were treated individually with 0.1%, 0.25% and 0.5% ( w / v ) colchicine solutions for 4, 6, 8, and 12 h. The colchicine-treated shoot tips were then inoculated on Murashige and Skoog (MS) medium fortified with 1.5 mg/l meta -Topolin for multiple shoot proliferation and later transferred into 1.5 mg/l indole-3-acetic acid-fortified MS medium for rooting of shoots. The ploidy levels of the colchicine-treated and regenerated plantlets along with the non-treated ones were confirmed via flow cytometry analysis and metaphasic chromosome count. The highest frequency of tetraploid plantlets (50%) were obtained when shoot tips were treated with 0.1% colchicine for 4 h. Morphological observations revealed that induced tetraploid plantlets exhibited delayed fresh shoot initiation, fewer but longer shoots, as well as fewer but broader leaves. Likewise, the study of stomata revealed that in comparison to their diploid counterparts, the tetraploid plantlets exhibited less frequent yet significantly larger stomata, and higher number of chloroplasts. The tetraploids were recorded with significantly higher chlorophyll, carotenoid, and anthocyanin content during the photosynthetic pigment analyses. During ex vitro acclimatization and field growth, the tetraploid plants exhibited delayed proliferation but with higher vigor and thickened broad leaves. The genetic uniformity among the diploid and the tetraploid plants was confirmed using conserved DNA-derived polymorphism (CDDP), directed amplification of minisatellite-region DNA (DAMD), inter simple sequence repeats (ISSR), and start codon targeted (SCoT) polymorphism marker systems. The tetraploids developed in the present study would be of immense importance for the genetic improvement of gerbera as far as its ornamental values are concerned., Competing Interests: Conflict of interestThe authors have no relevant financial or non-financial interests to disclose., (© King Abdulaziz City for Science and Technology 2023, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)

Background: Codonopsis pilosula (Franch.) Nannf. is a medicinal plant traditionally used in China, Korea, and Japan to treat many diseases including poor gastrointestinal function, low immunity, gastric ulcers, and chronic gastritis. The increasing therapeutic and preventive use of C. pilosula has subsequently led to depletion of the natural populations of this species thus necessitating propagation of this important medicinal plant. Here, we developed an efficient and effective in vitro propagation protocol for C. pilosula using apical shoot segments. We tested various plant tissue culture media for the growth of C. pilosula and evaluated the effects of plant growth regulators on the shoot proliferation and rooting of regenerated C. pilosula plants. Furthermore, the tissues (roots and shoots) of maternal and in vitro-regenerated C. pilosula plants were subjected to Fourier-transform near-infrared (FT-NIR) spectrometry, Gas chromatography-mass spectrometry (GC-MS), and their total flavonoids, phenolics, and antioxidant capacity were determined and compared., Results: Full-strength Murashige and Skoog (MS) medium augmented with vitamins and benzylaminopurine (1.5 mg·L -1 ) regenerated the highest shoot number (12 ± 0.46) per explant. MS medium augmented with indole-3-acetic acid (1.0 mg·L -1 ) produced the highest root number (9 ± 0.89) and maximum root length (20.88 ± 1.48 mm) from regenerated C. pilosula shoots. The survival rate of in vitro-regenerated C. pilosula plants was 94.00% after acclimatization. The maternal and in vitro-regenerated C. pilosula plant tissues showed similar FT-NIR spectra, total phenolics, total flavonoids, phytochemical composition, and antioxidant activity. Randomly amplified polymorphic DNA (RAPD) test confirmed the genetic fidelity of regenerated C. pilosula plants., Conclusions: The proposed in vitro propagation protocol may be useful for the rapid mass multiplication and production of high quality C. pilosula as well as for germplasm preservation to ensure sustainable supply amidst the ever-increasing demand., (© 2023. The Author(s).)

Exacum bicolor, an endemic and endangered medicinal plant, belongs to the family Gentianaceae. A rapid protocol has been developed for efficient multiple shoot induction by testing nodal explants on Murashige and Skoog (MS) medium supplemented with various cytokinins. The cytokinins 6-benzyladenine (BA), 6-furfurylaminopurine (Kn), 2-isopentenyladenine (2-iP) and zeatin (Zn) were used individually and in combination at different concentrations (0.5, 1.0, 2.0, 5.0, and 10.0 μM). The maximum number of shoots (19.33 ± 1.09 per explant) as well as their fresh weight (5.1 ± 0.68 g) and dry weight (216.83 ± 2.84 mg) were obtained with 10.0 μM BA + 2.0 μM Kn. After 4 wk, the multiple shoots from agar culture were subcultured into liquid medium containing the same growth regulator combinations. After 8 wk of liquid culture, the best treatment had about tenfold increase in shoot number (199.5 ± 1.14 per explant). The fresh weight (13.76 ± 0.14 g) and dry weight (909.33 ± 1.92 mg) were highest with full strength MS medium supplemented with 3% sucrose and containing 10.0 μM BA + 2.0 μM Kn. Maximum root development was observed after 30 d with 0.5 μM indole-3-butyric acid (IBA) supplementation. Regenerated plants were successfully transferred to pots containing coco peat:perlite mixture and showed a 75% survival rate. Genetic fidelity of in vitro plantlets compared to mother plant were assessed using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR) markers. All the regenerated plants were genetically identical to their mother plant, showing no detectable genetic variation in the regenerated plantlets. Thus, this protocol could be successfully used for mass multiplication and germplasm conservation of E. bicolor. [ABSTRACT FROM AUTHOR]

Sixty six plants raised via direct shoot bud organogenesis from pre-plasmolysed leaf explants of Catharanthus roseus were assessed under in vivo conditions for their physio-morpho traits, tryptophan metabolism, genetic fidelity and alkaloid profile. Morphologically all plants were parent like except the three morpho-types i.e. broad cup-shaped leaves, dwarf phenotype and enlarged pink corona. Vindoline was detected in most of the plants in substantial amount while ajmalicine, ajmaline and catharanthine were detected in few plants only. More than eightfold increased vindoline (0.08 % dry wt.) was showed by plant no. 1, 4 and 39. Except plant no. 54, which accumulated the highest alkaloid content of 3.09 % dry wt. with tryptophan content of 0.0283 % dry wt., majority of the plants showed a negative correlation between total alkaloid content and tryptophan accumulation and strong positive correlation between tryptophan content and 5-methyltryptophan tolerance. ISSR and RAPD profile of seventeen randomly selected, field established plants was generated by using 10 ISSR and 60 RAPD primers. In RAPD, a total of 753 bands were detected, out of which 624 (82.87 %) were monomorphic. In ISSR profiling, a total of 205 bands were detected out of which 200 bands were monomorphic (97.56 %) and only 5 bands were found to be polymorphic. Highly significant data for the monomorphic banding pattern across all the three genotypes ( p < 0.01) was observed. Principal components, AMOVA analysis and multivariate analysis using Nei and Li's coefficient further validate the monomorphism. Thirteen plants having superior traits were grown in Random Block Design in field. The relevance of physiological and epigenetic changes occurred in the light of morpho-types and alkaloid profile of directly regenerated plants during in vitro to in vivo acclimatization in C. roseus is discussed. [ABSTRACT FROM AUTHOR]

Genetic integrity of tissue culture-raised plantlets of broccoli cv. Solan green head from India was assessed using RAPD. First, highly efficient, reliable and high-frequency shoot regeneration was achieved in leaf (62.96%) and petiole (91.11%) explants on MS medium containing 4.5 mg/l BAP + 0.019 mg/l NAA, and 4.0 mg/l BAP and 0.5 mg/l NAA respectively. Maximum rooting ability (93.99%) with healthy and vigorous roots was observed on MS medium containing 0.20 mg/l NAA. The regenerated plantlets with well-developed shoot and root system were acclimatized successfully. For genetic stability studies, a total of 66 amplicons were amplified using 15 informative primers with a high degree of monomorphism (88.45%) across the mother plant and 20 randomly selected in vitro regenerated plantlets. [ABSTRACT FROM AUTHOR]

Tacca leontopetaloides is a tuberous species with nutritional, medicinal, and industrial potential. The aim of the present study was to develop a simplified micropropagation protocol for this species, combining phases of in vitro propagation and rooting, and to assess the genetic stability of in vitro regenerants using ISSR and flow cytometric analysis. In the first step, the effect of four cytokinins (BA, zeatin, kinetin, thidiazuron) at concentrations of 0.1-0.7 mg l-1 on shoot organogenesis was tested. The highest number of shoots was achieved on MS medium supplemented with 0.1 mg l-1 zeatin (4.11 ± 0.35 shoots/explant). Thereafter, this treatment was combined with auxins (NAA, IAA) at concentrations of 0.01-0.07 mg l-1 to study shoot organogenesis and in vitro rooting. A combination of plant growth regulators provided unchanged (high) shoot organogenesis, while the root number per explant increased. The highest number of roots of all tested treatments was produced on MS medium supplemented with zeatin (0.1 mg l-1) in combination with NAA (0.05 mg l-1) (5.00 ± 0.38 roots/explant), suggesting that this medium may be used for both propagation and rooting. Well-rooted plants were transferred ex vitro, with a 97.78% survival rate. Ten randomly chosen in vitro regenerants were subjected to ISSR and flow cytometric analysis. Eleven ISSR primers produced 40 clear and reproducible bands per sample. All amplified products were monomorphic; no polymorphism was detected. Similarly, the results of flow cytometric analysis showed no variation in ploidy level. Thus, the micropropagation method optimized here can be used for highly effective production of true-to-type plants of T. leontopetaloides. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The escalating loss of biological diversity throughout the world has become a major concern for the conservation biologists. Like other threatened plant species, the natural populations of the orchids are also severely threatened. Dendrobium thyrsiflorum is one such representative of the family Orchidaceae whose natural populations are getting destroyed at an alarming rate and deserves special conservation attention. Both direct shoot organogenesis (DSO) and indirect shoot organogensesis (ISO) pathways were experimented and the highest regeneration frequency for DSO and ISO pathways were found to be 86.2 and 96 % respectively. The regenerated shoots were best rooted in half-strength MS medium supplemented with 1 mg/l indole butyric acid (IBA) and 0.5 mg/l phloroglucinol. The genetic stability of the acclimatized plants derived from ISO and DSO pathways was assessed using Inter Simple Sequence Repeats (ISSR) and Start Codon Targeted (SCoT) molecular markers. SCoT proved to be a superior marker over ISSR in detecting clonal variability. The phytochemical analysis of the micropropagated plants also revealed a comprehensively higher yield of various secondary metabolites with significantly higher antioxidant potentials in both ISO- and DSO-derived plants over the mother plant. However, the ISO-derived plants were more phytochemically enriched compared to the DSO-plants. The rapid multiplication rate, higher genetic stability and secondary metabolite production ensures the utility of this micropropagation method for D. thyrsiflorum in the ex situ conservation and commercial exploitation of other important orchid species. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]