An efficient and reproducible in vitro plant regeneration protocol has been developed for Annona reticulata L., a medicinally important plant of family Annonaceae. This is the first report of an efficient in vitro micropropagation and genetic fidelity evaluation of Annona reticulata using nodal segments as explants. Nodal explants were cultured on Murashige and Skoog (MS) medium augmented with various concentrations of cytokinins alone and in combination with auxins. MS medium supplemented with 2.5 mg/l 6-benzylaminopurine (BAP) and 0.3 mg/l indole-3-butyricacid (IBA) combination has proven the best for optimum regeneration response (87.4%) and a maximum number of multiple shoots (6.33 ± 0.37). Rhyzogenesis occurred on half-strength MS medium supplemented with 4 mg/l IBA, resulted in the highest percentage of rooting (73%) and a maximum number of roots (4.65 ± 0.23) along with maximum root length (1.76 ± 0.08 cm). The well developed regenerates were shifted to the mixture of soil, sand and organic manure (2:1:1) in small plastic cups and acclimatized successfully. Genetic fidelity of the discussed etiquette was validated using two types of DNA fingerprinting techniques i.e. start codon targeted (SCoT), and inter simple sequence repeat (ISSR) analyses. Among the ten SCoT (SC) and ISSR primers used, an excellent amplification with scorable DNA bands was produced by SC9 and ISSR4 primers. The results showed that the regenerated plantlets are monomorphic and true-to-type with mother plant. This study provides important information about selection of suitable regeneration medium to improve multiple shoot induction and rooting of Annona reticulata. It can be used for commercial cultivation and for further genetic redevelopment investigation studies, also applied for large scale propagation of elite genotypes.

In vitro micropropagation and genetic fidelity studies using SCoT and ISSR primers in Annona reticulata L.: an important medicinal plant

Organogenesis and evaluation of genetic homogeneity through SCoT and ISSR markers in Helicteres isora L., a medicinally important tree.

The present investigation was carried out to for developing a competent protocol for micropropagation of commercial plum cultivar Frontier, through control of shoot tip necrosis (STN) observed during in vitro shoot multiplication. Apical and axillary buds were collected from elite high yielding tree of Frontier and inoculated on Murashige and Skoog (MS) medium fortified with phytohormones at different concentrations. MS medium containing 0.5 µM BA, 0.5 µM Kin and 0.2 µM IBA resulted in highest shoot bud establishment (58.95%) after 4 weeks interval. However, all the shoots showed necrosis and died after 1st subculture. Necrosis appeared with yellowing of shoot tips within 7 days of first subculture and gradually increased downwards resulting in yellowing of leaves, followed by death of the shoots. Among various reported methods tested to control STN, combination of Fructose (0.50 mM) and Calcium chloride (1.0 mM) proved 100% effective. Highest in vitro shoot multiplication (fivefold) was recorded on medium enriched with 2.0 µM BA, 0.2 µM GA3, 0.2 µM IBA, 0.50 mM Fructose and 1.0 mM Calcium chloride. For rooting, two step rooting procedure was followed which resulted in 85.00% rooting when microshoots were given a dark treatment by dipping in ½ MS broth supplemented with 2.5 µM IBA for 48 h before transferring to semisolid ½ MS basal medium. Rooted plantlets when transferred in vivo for acclimatization, showed 70–80% survival after 10 weeks and no mortality on field transfer. Molecular analysis of the plants using ISSR primers proved 100% similarity between them and mother tree.

Propagation of plum (Prunus salicina L.) cultivar Frontier in vitro through control of shoot tip necrosis (STN) and validation of genetic integrity using ISSR markers

BACKGROUND AND OBJECTIVE Jojoba is a promising industrial plant, which recommended with pharmaceutical benefits. The present study was conducted to stimulate embryogenic calli formation from jojoba using zeatin and thidiazuron (TDZ), as well as determination of the antioxidant activity of proliferated calli. MATERIALS AND METHODS For callus induction, leaf and stem explants derived from in vitro grown shootlets, were cultured on Murashige and Skoog (MS) medium with different combinations of 0.5 mg L-1 benzyl adenine (BA) or kinetin with 2,4-Dichlorophenoxyacetic acid (2,4-D), Naphthalene acetic acid (NAA) and picloram at 0.5 or 1mg L-1. To stimulate embryogenic calli, friable callus were transferred to woody plant medium (WPM) supplemented with different concentrations of zeatin or TDZ. Antioxidant activity of different treatments was determined using hexane or petroleum ether extraction. Data was analyzed as mean±standard deviation (SD). RESULTS The MS medium supplemented with 0.5 mg L-1 BA+0.5 or 1 mg L-1 picloram was the best treatment to obtain friable calli from both explants types. WPM medium supplemented with 2 mg L-1 zeatin gave the highest percentage of embryogenic calli derived from leaf explants. While the highest percentage of embryogenic calli derived from stem explants was registered using 1 or 4 mg L-1 TDZ containing medium. Embryogenic calli originated from leaves explants on 1.5 mg L-1 zeatin showed promising activity of antioxidant with hexane extraction. However, embryogenic calli originated from stem explants on 1 mg L-1 TDZ showed the highest antioxidant activity with petroleum ether extraction. CONCLUSION TDZ has promising effect on embryogenic callus induction from stem explants. While, zeatin has promising effect on embryogenic callus induction from leaf explants.

Zeatin and Thidiazuron Induced Embryogenic Calli From In Vitro Leaf and Stem of Jojoba (Simmondsia chinensis).

Micropropagation has an important role in the large-scale production of blackberry plant material, given the high proliferation rates of this species. The aim of the present study was to evaluate the proliferative capacity of blackberry grown in vitro on wheat starch-gelled culture medium compared to classical agar-gelled medium and to assess the genetic fidelity between the proliferated shoots in starch-gelled culture medium and their mother plants. Six blackberry varieties (‘Čačanska Bestrna’, ‘Chester Thornless’, ‘Driscoll’s Victoria’, ‘Loch Ness’, ‘Polar’, and ‘Karaka Black’) were tested. For the in vitro shoots proliferation, Murashige and Skoog (MS) medium supplemented with 0.5 mg dm−3 6-benzyladenine (BA) was used. The conventional medium was gelled with 0.5% plant agar, and wheat starch was used as an alternative gelling agent in a concentration of 5%. The results showed that for all blackberry cultivars, the highest number of shoots/inoculum was obtained in wheat starch-gelled culture medium, with a maximum value of 54.42 ± 4.18 presented by ‘Karaka Black’. Considering the length of the proliferated shoots, all tested cultivars presented outstanding results on the culture medium gelled with 5% wheat starch. The highest values regarding shoots length were observed on the ‘Chester Thornless’ followed by ‘Čačanska Bestrna’, and ‘Loch Ness’ with values of 5.55 ± 0.04 cm, 5.46 ± 0.06 cm, and 5.37 ± 0.09 cm, respectively. The genetic uniformity of the micropropagated shoots in relation to their mother plants was confirmed by sequence-related amplified polymorphism (SRAP) and start codon targeted (SCoT) molecular markers. 

The Use of Wheat Starch as Gelling Agent for In Vitro Proliferation of Blackberry (Rubus fruticosus L.) Cultivars and the Evaluation of Genetic Fidelity after Repeated Subcultures

This study is aimed at developing an efficient method for micropropagation of true-to-type jojoba plants. Nodal segments were used for in vitro shoots proliferation. Among three concentrations of BA used for multiplication, 1 mg/l BA gave the highest number of shoots. To enhance growth of shoots, combinations of BA and Kn were investigated. The greater value of shoot length and the maximum number of nodes were observed in the medium containing 1 mg/l BA + 1.5 mg/l Kn. Among different medium used to increase the rate of multiplication, the maximum number of shoots was recorded at ½ MS + Gamborg B5 (B5) vitamins + 2 mg/l Kn + 10 mg/l adenine sulphate (AS). Rooting was obtained upon supplementation of ½ Woody Plant Medium (WPM) with 1 mg/l IBA. Acclimation was achieved by transplanting rooted plantlets into pots containing peat moss and vermiculite . Start codon targeted (SCoT) and intersimple sequence repeat (ISSR) analyses were carried out to assess the genetic fidelity of micropropagated plantlets. All banding profiles of the two analyses from micropropagated plants were monomorphic and similar to the mother plant. Hence, this protocol can be employed for commercial micropropagation of jojoba without the risk of losing genetic stability. Plant Tissue Cult. & Biotech. 25(2): 165-179, 2015 (December)

/pdf/micropropagation-and-assessment-of-genetic-stability-of-in-4owgkmtkyn.pdf

Micropropagation and Assessment of Genetic Stability of In Vitro Raised Jojoba (Simmondsia chinensis Link.) Plants Using SCoT and ISSR Markers

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Micropropagation and Assessment of Genetic Stability of In Vitro Raised Jojoba (Simmondsia chinensis Link.) Plants Using SCoT and ISSR Markers