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Solena amplexicaulis (Lam.) Gandhi is a creeping cucurbit with high medicinal values in terms of its therapeutic uses. Traditionally, this plant species is used as a folklore medicine for the treatment of various disorders like spermatorrhea, urinary retention, jaundice, and cardiovascular disorders. Owing to high medicinal importance, it is overexploited, which has resulted in decrease in its population size in the wild. Furthermore, its natural propagation through root tubers and seeds is limited. In view of this constraint, the present study of in vitro micropropagation was chosen as an alternative for its propagation and conservation. Murashige and Skoog (MS) media amended with plant growth regulators like 6-benzylaminopurine (BAP) and thidiazuron (TDZ) independently and with a combination of indole-3-acetic acid (IAA) were tested for in vitro shoot induction by using nodal explants. The highest number of shoots (32.84±0.02) per explant was achieved on Murashige and Skoog (MS) medium with 1.0 mg L-1 TDZ and 1.5 mg L-1 IAA. The induced shoots were elongated with a maximum shoot length of 10.25±0.01 cm on MS medium augmented with 2.0 mg L-1 gibberellin (GA3) and 1.5 mg L-1 TDZ. Rooting of in vitro raised shoots was attained on half-strength MS medium fortified with 1.0 mg L-1 indole-3-butyric acid (IBA) and 2.0% sucrose. The hardening of resultant complete plantlets was carried out in plastic cups by using a 2:1 ratio of sterile black soil and sand; initially, they were kept in greenhouse conditions and subsequently shifted to natural field conditions with 78% of survival rate. The genetic fidelity of tissue-cultured plants was confirmed by ISSR (inter simple sequence repeats) and SCoT (starting codon targeted) primer-based analysis. [ABSTRACT FROM AUTHOR]

Artemisia vulgaris is a medicinally important essential oil–yielding plant belonging to Asteraceae, used as a traditional remedy against chronic diseases. The essential oil of this plant has been used as an insect repellent and antimicrobial agent. The growing concern of antibiotic resistance has forced to find an alternative way to control multi-drug-resistant urinary tract–infecting bacterial pathogens by using the essential oil and leaf extract of A. vulgaris for the first time. The antibacterial activity showed that the essential oil (in situ, ex vitro) and leaf crude extract (ex vitro) are highly effective on all human urinary tract–infecting pathogens. In contrast, ex vitro plant-mediated essential oil is most significant with an 18.07 ± 0.17-mm inhibition zone against Klebsiella pneumoniae. The ex vitro plants are also superior to the in situ plants for obtaining greater extract and essential oil content (950 µl), over a time period of 12 wk. To obtain the ex vitro plants, meta-topolin is used for in vitro regeneration of A. vulgaris for the first time, showing the highest mean shoot number (98.11 ± 0.31) regeneration after 42 d, which is more significant in comparison with other studies conducted till date. The cytogenetic stability of the regenerated plantlets has been checked using start codon targeted polymorphism and cytological studies, conferring the homogeneity among regenerants along with in situ plant. Furthermore, the essential oil of the ex vitro plants was analysed through gas-chromatography mass spectroscopy, which detected a few compounds with bio-significance from A. vulgaris, including 2-carene, 2-(4-nitrophenyl) acetamide, β-guaiene, α-acorenol, and 10,12-Tricosadiyonic. [ABSTRACT FROM AUTHOR]

Teucrium (Teucrium polium L.) is one of the valuable medicinal plants, but its poor seed germination is one of the principal problems of its extensive cultivation. Somatic embryogenesis is crucial for medicinal plant genetic improvement, germplasm preservation, and in vitro mass propagation. Synthetic seed production is a suitable alternative for natural seed production in biotechnology and plant development. The objectives of the present study were to optimize somatic embryogenesis, produce synthetic seeds, and evaluate the genetic fidelity of the regenerated T. polium. Results suggested that MS medium supplemented with 1.0 mg L−1 kinetin (Kin) alone and without auxin demonstrated the maximum embryogenesis and regeneration rates in leaf and petiole explants. The simultaneous occurrence of several somatic embryonic developmental stages, such as the globular, heart-shaped, torpedo-shaped, and cotyledonary stages, on an embryogenic callus clearly suggested that T. polium embryogenesis accurately captures an asynchronous process. The petiole and leaf explants cultured on MS medium with 1.0 mg L−1 Kin showed the highest rates of plant regeneration with spontaneously root development. Compared to somatic embryos, synthetic seeds with meristem sources lacked callogenesis. At 25 °C for 4 wk and at 4 °C for 3 wk, the meristem source caused the highest percentage of synthetic seeds to germinate (96.66%), while − 18 °C had a negative impact on the germination of the synthetic seeds. The results suggested that there was no genetic variation identified in the ISSR analysis on in vitro–generated T. polium plants. [ABSTRACT FROM AUTHOR]

Indian tea germplasm is known for its diversity, which not only assists the development of superior planting material for tea breeding but also functions as a genetic resource for other tea-producing countries. In this study, the aim was to develop an improved cryo-protocol for long-term conservation of tea germplasm. Shoot tips (approximately 2 mm) of Camellia sinensis were cryopreserved using vitrification and V cryo-plate techniques. The recovery rate of cryopreserved shoot tips obtained using vitrification was 33.2% at 90 min of Plant Vitrification Solution 2 (PVS2) exposure. To improve recovery rates and tolerance to cryo-injury, shoot tip donor plants were preconditioned with cold hardening, high sucrose, abscisic acid (ABA), and proline prior to cryopreservation. The vitrification technique resulted in an increase of regrowth of shoot tips to 60.5% after preconditioning of shoot tip donor plants for 30 d at 25 °C with 10 mg L−1 ABA and 3 mg L−1 proline. Regrowth further increased to 75.6% with the V cryo-plate technique using shoot tip donor plants preconditioned with the above concentrations of ABA and proline. After cryopreservation, shoot tips were recovered on regrowth medium that developed into shoot cultures in 5 wk. Random amplified polymorphic DNA (RAPD) analysis was performed to test genetic fidelity after cryopreservation and results showed 100% monomorphism among all the regenerants and 10-mo-old in vitro mother cultures. Results showed that the preconditioning strategies along with V cryo-plate technique influenced and improved the cryo-recovery rate of shoot tips of tea and provided a robust protocol for tea cryopreservation. [ABSTRACT FROM AUTHOR]

The present study was focused towards determining the factors governing direct organogenesis in kiwifruit cultivar 'Allison'. Using in vivo buds or nodal segments as explants, the maximum 75.5% uncontaminated explants were obtained upon consecutive individual disinfection treatment with 1% carbendazim (3 min), 0.1% HgCl2 (1 min), and 2% NaOCl (2 min), respectively. The maximum shoot regeneration response of 84.81% was recorded on Murashige and Skoog (MS) medium fortified with 0.45 μM BA, 0.1 μM IBA, and 0.13 μM GA3. The months of May displayed the highest in vitro explant regeneration response for two consecutive years. The highest shoot proliferation rate was 1:6 with a 5.34 mean number of shoots and a maximum mean shoot length of 6.32 cm on MS medium containing 0.22 μM BA, 0.1 μM IBA, and0.69 μM GA3. A two-step or sequential approach for in vitro root induction showed good quality rooting response of 91.03% producing a higher number of long (2.23 cm) roots per shoot, and afterwards, the plantlets were hardened successfully with a higher survival rate (95%). Also, ISSR markers confirmed the genetic fidelity of the in vitro-raised regenerants. [ABSTRACT FROM AUTHOR]

Zingiberaceae family, also known as the ginger family, comprises 53 genera and 1300 species. The members of this family are widely known for their ethnobotanical properties. Owing to their anti-inflammatory, antimicrobial, antidiabetic, and cardioprotective properties, the rhizomes have been used in traditional medicine to treat a wide range of illnesses like cold, inflammation, gastritis, arthritis, diabetes, and respiratory problems. The medicinal properties of the plants are attributed to the wide array of secondary metabolites present in them. They also find their use in food, spices, ornamentals, perfumes, and cosmetics. The current review focuses on biotechnological techniques like direct regeneration, indirect regeneration via callus induction and somatic embryogenesis, microrhizome induction, and in vitro secondary metabolite production that have been established to meet the increasing commercial and pharmaceutical demands. This review also identifies unrevealed possibilities, knowledge gaps, and considerable challenges that should be taken into account for developing novel concepts pertaining to the biotechnology of Zingiberaceae in the near future. [ABSTRACT FROM AUTHOR]

Eulophia dabia (D. Don) Hochr is an endemic, critically endangered, terrestrial orchid species having profound importance in traditional herbal drug preparations and pharmacopoeia worldwide. In the present investigation, we have developed an efficient and genetically stable micropropagation protocol of E. dabia using axenic rhizome as explant. Seeds inoculated on half-strength Murashige and Skoog (MS) medium showed 5% germination efficiency. Scanning electron microscopy analysis revealed differential effects of various sterilizing agents (NaOCl2 and ethanol) on the seed coat integrity leading to a significant improvement in seed germination rate. The rhizome-like bodies (RLBs) developed after 6 mo of seed germination showed best growth of rhizome (size: 3.3 × 1.5 cm and 5.0 nodes per rhizome) in MS medium containing casein hydrolysate and activated charcoal. One-year-old mature axenic rhizomes were further sub-cultured for multiple shoot induction, and maximum 96.1% response was observed in MS medium fortified with 4.4 μM BAP and activated charcoal with 4.3 mean shoot number and 13.4 cm shoot length. After 3 to 4 sub-cultures using the same medium, bipolar structures (plantlets with shoot and root) were developed. The properly developed plantlets were successfully acclimatized to open environment with 98% survival. Genetic stability of the discussed protocol was evaluated by random amplified polymorphic DNA (RAPD) markers, which proved true to typesets of the in vitro raised plants. In the present study we optimized in vitro propagation system for the E. dabia which can be successfully applied further for the genetic improvement studies and commercial scale propagation. [ABSTRACT FROM AUTHOR]

Micropropagation technique has been recommended for plant tissue rejuvenation/reinvigoration and the improvement of clonal plants production commercially. Concerning the limiting factors on in vitro cultivation of Eucalyptus grandis × E. urophylla hybrid clone, the effects of light intensity on in vitro introduction and multiplication phases were assessed. The experimental tissue was collected from ministumps cultivated in a semi-hydroponic system. The results of light intensity on in vitro introduction and multiplication were evaluated with four treatments (fluorescent lamp/40 µmol m−2 s−1, red/blue LEDs 20 µmol m−2 s−1, red/blue LEDs 40 µmol m−2 s−1, and red/blue LEDs 80 µmol m−2 s−1). The explants' morphological, anatomical, and genetic characteristics were evaluated at 30 d in the in vitro introduction, and 12 subcultures for in vitro multiplication. Based on the results, the treatments F/L 40 µmol m−2 s−1 and R/B 80 µmol m−2 s−1 are the most suitable during in vitro introduction and multiplication of Eucalyptus grandis × E. urophylla. It provides a higher percentage of responsive explants, shoots per explant, vigor, photosynthetic pigment content, adaxial epidermis thickness, abaxial epidermis, mesophyll, palisade parenchyma, spongy parenchyma, and vascular tissues. Furthermore, it was found that the micropropagated plants are identical clones of the Eucalyptus grandis × E. urophylla selected tree; that is, there was no somaclonal variation during the twelve subcultures on in vitro multiplication. [ABSTRACT FROM AUTHOR]

A highly efficient and reproducible in vitro plant regeneration method has been developed from shoot bud, half-shoot, and shoot slice explants of four genotypes of turmeric (Curcuma longa L.). The shoot slice explant produced the highest number of shoots (22.4 shoots explant−1) than half-shoot (18.6 shoots explant−1) and shoot bud explant (14.2 shoots explant−1) of Salem genotype on medium amended with 13.32 µM 6-benzylaminopurine (BAP) and 5.37 µM 1-naphthaleneacetic acid (NAA). Salem genotype was more efficient among the four genotypes tested, followed by Duggirala Red, Prathibha, and PCT-13 genotypes. Approximately 80% of shoots were induced spontaneous rooting on shoot induction media fortified with NAA. The complete plantlets were obtained after rooting the shoots on a medium augmented with 4.90 µM Indole-3-butyric acid (IBA). IBA was an efficient auxin to induce the maximum number of roots per shoot than Indole-3-acetic acid (IAA) and NAA. The plantlets were acclimatized under greenhouse conditions, and the survival rate was recorded as 96%. The plantlets obtained from all explants were morphologically similar to mother plants. Two genome-based molecular markers, namely inter simple sequence repeats (ISSR) and start codon targeted (SCoT) molecular markers, were employed to determine the genetic stability of plants obtained from the tissue culture of the Salem genotype. The DNA markers validated the genetic uniformity of the plants obtained from all three explants was similar to their mother plant. This plant regeneration procedure could be a helpful method for the genetic improvement of turmeric. [ABSTRACT FROM AUTHOR]

Scaevola taccada (Gaertn.) Roxb. is a hemi-sclerophyllous littoral shrub of the family Goodeniaceae. It is a salt-tolerant plant and used in soil reclamation and coastal landscaping programs to control beach erosion. The present article reports a reproducible in vitro regeneration system using meta-topolin (mT) through liquid medium employing nodal segment cultures. The highest number of axillary shoots (5.8 ± 0.29) was differentiated on Murashige and Skoog's (MS) medium containing meta-topolin (1.5 mg L−1). The amplification of shoots (49.8 ± 0.37) and fresh growth was achieved by recurrent subcultures on liquid MS medium supplemented with 0.25 mg L−1mT and 0.15 mg L−1 indole-3-acetic acid (IAA) in the presence of additives (50 mg L−1 ascorbic acid + 25 mg L−1 citric acid, adenine sulphate and l-arginine). Rooting and acclimatization was simultaneously achieved using concurrent ex vitro rooting and acclimatization (CEVRA) technique by pulse treating the shoot base with 300 mg L−1 IBA for 5 min. The in vitro developed shoots represented a maximum percentage (100%) of rooting in autoclaved soilrite in a greenhouse. After 2 mo, the plantlets were established in the field with cent percentage survival success. The genetic fidelity analysis using inter simple sequence repeats (ISSR) DNA molecular markers developed maximum of 55 monomorphic bands and revealed the genetic homogeneity of in vitro regenerated plantlets with the mother plant. [ABSTRACT FROM AUTHOR]

An in vitro regeneration system for propagation of a valuable medicinal tree, Pterocarpus marsupium, using shoot tip (ST) explants derived from 7-d-old axenic seedlings has been successfully developed. Murashige and Skoog (MS) medium containing cytokinins (BA, mT, or Kn) and auxins (NAA, IAA) in different concentrations and combinations showed to have a marked stimulatory effect on the regeneration output. Of cytokinins, meta-topolin (mT 7.0 μM) proved optimum dose for multiple shoot induction yielding 6.50 ± 0.49 shoots per explant with mean shoot length (4.00 ± 0.16 cm) in 70% cultures after 6 wk. Whereas, supplementation of low concentration of auxin (1.0 μM NAA) with optimal cytokinin (7.0 μM mT) favored enhanced shoot induction with increased percent response. At this level, a maximum of 13.54 ± 0.34 shoots per explant with (5.30 ± 0.10 cm) mean shoot length were recorded in 80% cultures after 12 wk. Thereafter, microshoots were isolated and their basal end treated with high doses of indole-3-butyric acid (250 μM) for 5 d followed to transplant onto various potting substrates for ex vitro root formation. A maximum of 3.63 ± 0.0.08 roots per microshoot and mean root length of 3.59 ± 0.07 cm with highest rooting frequency (67.7%) was observed after 4 wk of transplantation. During ex vitro rooting-cum-acclimatization, various physiological parameters were found to be fluctuating initially for 35 d; thereafter, an increasing trend was observed in net photosynthetic rate, intercellular CO2 concentration, and chlorophyll a/b estimation. Overall, the successfully acclimatized regenerants when transferred in natural condition showed about 96.7% survival rate after 3 mo. Assessment of genetic integrity of tissue culture raised plantlets was accomplished by DNA–based ISSR primers. A total of 35 bands with an average of 4.38 bands per primer were scored in monomorphic banding pattern. Thus, it is apparent that the mode of in vitro regeneration protocol was suitable for obtaining true-to-type and disease-free plants. [ABSTRACT FROM AUTHOR]

The present study describes the first successful report on an efficient in vitro protocol for direct and indirect organogenesis of an important medicinal and anticancerous plant, Solanum erianthum D. Don. An optimal response using leaf explants was achieved in half-strength Murashige and Skoog (MS)-medium supplemented with 5 mg L−1 naphthalene acetic acid for friable callus formation and 2 mg L−1 naphthaleneacetic acid and 0.5 mg L−1 6-benzylaminopurine amino purine for induction of compact callus. The maximum number of shoot buds (20.33 ± 11.55) was obtained from compact callus using indirect shoot organogenesis in MS medium supplemented with 0.5 mg L−1 thidiazuron among the four cytokinins tested, which included 6-benzyl amino purine, kinetin, 6-γ,γ-dimethylallylamino purine, and thidiazuron. Supplements of 3 mg L−1 6-benzyl amino purine played a crucial role in direct shoot bud formation from a leaf. The buds appeared as small protuberances, and finally developed into leafy shoots as the culture period progressed. Regenerated shoot buds were transferred to MS media that contained four cytokinins for shoot tip culture, in which 8 mg L−1 6-γ,γ-dimethylallylamino purine exhibited the best response (15 ± 2.52). Although efficient plant regeneration was achieved in the presence of all four tested cytokinins in the three organogenetic pathways, distinct differences exist in terms of shoot regeneration potential. Half-strength MS basal medium is recommended for root induction. An approximate 70% survival rate of regenerated plantlets was recorded following sequential transfer from culture conditions to the field. The genetic fidelity of the regenerated plants was evaluated using random amplified polymorphic DNA molecular markers, which showed a high number of uniform monomorphic bands. [ABSTRACT FROM AUTHOR]

To enhance the multiplication rate in Musa acuminata Colla (banana; 'Grand Nain') organogenesis, higher amounts of thiamine along with different sugar types and concentrations were evaluated at the proliferation phase. Thiamine at 1, 10, 50, 100, and 200 mg L−1 was compared with 0.1 mg L−1 thiamine found in conventional Murashige and Skoog (MS) medium. Maximum proliferation of banana was induced with 100 mg L−1 thiamine. Additionally, 15, 30, and 45 g L−1 sucrose, glucose, fructose, and sorbitol combined with regular and optimal levels of thiamine were tested. Glucose at 30 g L−1 most improved shoot proliferation alone and enhanced shoot proliferation further, when combined with 100 mg L−1 thiamine, followed by sucrose and fructose, whereas sorbitol completely inhibited growth and caused tissue browning. All evaluated vegetative traits were significantly affected by sugar type and concentration, and thiamine levels, unlike the photosynthetic pigments. Moreover, genetic stability of the plants recovered from the enhanced protocol was confirmed by inter-simple sequence repeats (ISSR) and randomly amplified polymorphic DNA (RAPD) analysis. A total of 230 bands generated by both marker types were monomorphic for the randomly selected regenerated plants, compared with their mother plant. Thus, the proliferation medium supplemented with 30 g L−1 glucose and 100 mg L−1 thiamine could be recommended for banana organogenesis. Results herein are of great importance and helpful in enhancing the commercial in vitro propagation protocols of banana, without the need of increasing the number of subcultures, which can cause somaclonal variation. [ABSTRACT FROM AUTHOR]

Korean arbor vitae (KAV; Thuja koraiensis Nakai) is a critically endangered coniferous tree in Korea. Here, we report the somatic embryogenesis (SE) and cryopreservation system that can be used for micropropagation of KAV and long-term storage of KAV cultures. To induce SE in KAV, the influence of the developmental stage of zygotic embryos and the effect of basal medium on embryogenesis induction were examined. The developmental stage of zygotic embryos had a significant effect on the embryogenesis induction (P < 0.0001). The highest frequency of embryogenesis induction occurred in megagametophytes with zygotic embryos at precotyledonary (P) and late embryogeny (L1) stage (36%). The highest frequency of embryogenesis induction was obtained on initiation medium containing IM basal salts with 2.2 μM 6-benzylaminopurine and 4.5 μM 2,4-dichlorophenoxyacetic acid (35%). The effect of abscisic acid (ABA) on production of somatic embryos was tested. The highest number of somatic embryos per 50 mg of embryogenic tissue was achieved on maturation medium with levels of 100 μM ABA (24.0 ± 2.4). The effect of cryopreservation treatment to embryogenic tissues on the maturation capacity of somatic embryos was also tested. No significant differences between noncryopreservation and cryopreservation treatment were observed (P = 0.1896), and the highest mean number of somatic embryo per 50 mg of embryogenic tissues was obtained in noncryopreserved cell line (28.17 ± 5.66). Finally, the genetic identities of the plantlets regenerated from non- and cryopreserved embryogenic cell lines were verified and there was no genetic variation in the regenerated plantlets from cryostored embryogenic cell lines. This study is the first report on SE and the successful cryopreservation of embryogenic culture of the genus Thuja. [ABSTRACT FROM AUTHOR]

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]

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]

Questions often arise concerning the genetic stability of plant materials stored in liquid nitrogen for long time periods. This study examined the genetic stability of cryopreserved shoot tips of Rubus germplasm that were stored in liquid nitrogen for more than 12 yr, then rewarmed and regrown. We analyzed the genetic stability of Rubus grabowskii, two blackberry cultivars (“Hillemeyer” and ‘Silvan’), and one raspberry cultivar (“Mandarin”) as in vitro shoots and as field-grown plants. No morphological differences were observed in greenhouse-grown cryopreserved plants when compared to the control mother plants. In the field, cryopreserved plants appeared similar but were more vigorous than mother plants, with larger leaves, fruit, and seeds. Single sequence repeats (SSR) and amplified fragment length polymorphism (AFLP) analyses were performed on shoots immediately after recovery from cryopreservation and on shoots subcultured for 7 mo before analysis. Ten SSR primers developed from “Marion” and “Meeker” microsatellite-enriched libraries amplified one to 15 alleles per locus, with an average of seven alleles and a total of 70 alleles in the four genotypes tested. No SSR polymorphisms were observed between cryopreserved shoots and the corresponding mother plants regardless of subculture. Although no polymorphisms were detected in shoots analyzed immediately after recovery from cryopreservation, AFLP polymorphisms were detected in three of the four Rubus genotypes after they were subcultured for 7 mo. Field-grown plants from the polymorphic shoot tips of R. grabowskii and ‘Silvan’ displayed the same AFLP fingerprints as their corresponding mother plants. Only long-cultured in vitro shoot tips displayed polymorphisms in vitro, and they were no longer detected when the plants were grown ex vitro. The transitory nature of these polymorphisms should be carefully considered when monitoring for genetic stability. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]