In vitro mass propagation of Cannabis sativa L.: A protocol refinement using novel aromatic cytokinin meta-topolin and the assessment of eco-physiological, biochemical and genetic fidelity of micropropagated plants

Role of phytohormones in regulating cold stress tolerance: Physiological and molecular approaches for developing cold-smart crop plants

To evaluate genetic homogeneity of 1-year-old guava (Psidium guajava L.) plants developed from in vitro somatic embryogenesis, DNA from leaf tissues of seven randomly selected plants along with the mother plant, was isolated and subjected to molecular analysis. A total of six Simple Sequence Repeat (SSR) primer pairs, producing reproducible and clear bands ranging from 100 to 300 bp in size, resulted in amplification of single band (allele), corresponding homozygous individuals. Moreover, of 10 different inter-simple sequence repeat (ISSR) primers screened, six produced resolvable, reproducible and scorable bands. All these ISSRs produced a total of 25 bands, ranging between 300 and 1,200 bp length, and the number of scorable bands, for each primer varied from three to six with an average of 4.1 bands per primer. The amplification products were monomorphic across all the micropropagated plants produced by all SSR and ISSR primers applied. The monomorphic banding pattern in micropropagated plants and the mother plant confirms the genetic homogeneity of the in vitro raised plants and demonstrates the reliability of our in vitro propagation system for guava.

Genetic homogeneity of guava plants derived from somatic embryogenesis using SSR and ISSR markers

A reliable and reproducible protocol for in vitro regeneration has been developed for Alhagi maurorum, a rare and medicinally important plant of family fabaceae. MS medium with BAP (2.0 mg l−1) proved to be the best for shoot bud induction from nodal segments. The rate of shoot multiplication was found to be influenced by a number of factors, viz., media composition, plant growth regulator’s type and concentration, successive transfer of mother explant for different passage, culture vessels and gelling agents. Modified MS medium (modified having nitrates reduced to half) solidified with 0.14 % gelrite and containing BAP (0.5 mg l−1), IAA (0.1 mg l−1) and additives was found optimum for shoot multiplication which gave rise to maximum number of shoots (33.5 ± 3.43 per culture vessel). The in vitro regenerated shoots were rooted under both in vitro (on half strength MS salts with 1.0 mg l−1 IBA + 100 mg l−1 activated charcoal) as well as ex vitro (on sterile soilrite by treating shoot base with 250 mg l−1 each of IBA and NOA for 4 min in green house) conditions. Thereafter, the in vitro and ex vitro rooted plantlets were hardened under green house conditions with 70 and 90 % survival rate, respectively. Start codon targeted (SCoT), inter simple sequence repeats (ISSR) and random amplified polymorphic DNA (RAPD) markers were used to validate the genetic homogeneity of seven tissue cultured plantlets growing in green house condition with mother plant. The amplification products were monomorphic across all the seven micropropagated plants as well as mother plant produced by all SCoT, ISSR and RAPD primers applied. The monomorphic banding pattern in micropropagated plants and the mother plant confirms the genetic homogeneity of the in vitro raised plants and demonstrates the reliability of our in vitro propagation system for A. maurorum. To the best of our knowledge, this is the first report on micropropagation and genetic homogeneity assessment of A. maurorum, which can be applied for large scale multiplication of elite genotypes of A. maurorum.

Micropropagation and validation of genetic homogeneity of Alhagi maurorum using SCoT, ISSR and RAPD markers

Lawsonia inermis L. – A commercially important primaeval dying and medicinal plant with diverse pharmacological activity: A review

A micropropagation method for Jaal (Salvadora persica)—a tree of arid horticulture and forestry has been developed. Nodal segments of fresh shoot sprouts originated from axillary buds obtained from a plant around 35–40 years old lopped plant were used as explants for establishment of in vitro cultures. Surface-sterilized explants produced optimum number of shoots through activation of axillary buds on Murashige and Skoog’s (MS) medium containing 8.88 μM BA (6-benzyladenine) + additives (25 mgl−1 each of adenine sulphate, arginine, citric acid, 50 mgl−1 ascorbic acid). The shoot multiplication was influenced by the successive transfer of the mother explants for 4–5 passages. The maximum number (23.1 ± 0.73 shoots per explant) of shoots were regenerated on MS supplemented with 1.11 μM BA + 1.16 μM Kn (Kinetin) + 0.54 μM NAA (α-naphthalene acetic acid). About 90% shoots pulse-treated with a combination of 2460.27 μM Indole-3-butyric acid (IBA) + 494.56 μM NOA (2-naphthoxy acetic acid) were rooted ex vitro on soilrite within 15–18 days. Over 80% cloned plantlets were hardened successfully in a green house and transferred to polybag/pots.

Micropropagation of Salvadora persica-a tree of arid horticulture and forestry

In vitro propagation of Eulophia nuda Lindl., an endangered orchid

Lawsonia inermis Linn. (Mehandi) is cultivated as cash crop in India particularly in Sojat area of Pali district, Rajasthan. Present investigation describes an efficient regeneration system for elite genotype of L. inermis using nodal segments. Optimum response in terms of percent cultures responding, days to bud break and average shoot length was observed on MS medium supplemented with 6-benzylaminopurine (BA; 2.0 mg l−1). Shoot multiplication was influenced by plant growth regulators, repeated transfer of explants and addition of ammonium sulphate. Maximum shoots were regenerated on MS medium supplemented with BA (0.25 mg l−1), kinetin (Kn; 0.25 mg l−1), indole-3-acetic acid (IAA; 0.1 mg l−1) and ammonium sulphate (150 mg l−1). To reduce resources, time and labours costs, we have also attempted ex vitro rooting of shoots. About 95 % shoots were rooted ex vitro on soilrite after treatment with indole-3-butyric acid (IBA; 300 mg l−1) and 2-naphthoxy acetic acid (NOA; 100 mg l−1) and establishment in soil successfully.

/pdf/micropropagation-of-commercially-cultivated-henna-lawsonia-2pt46357ef.pdf

Micropropagation of commercially cultivated Henna (Lawsonia inermis) using nodal explants.

An in vitro propagation method for female plants of Momordica dioica (Roxb.) has been established. The nodal segments were harvested and the cut ends of the explants were sealed with wax and then surface sterilized and cultured. Bud breaking occurred on Murashige and Skoog’s (MS) agar-gelled medium + 2.0 mg L−1 6-Benzylaminopurine (BAP) + 0.1 mg L−1 Indole-3 acetic acid (IAA). The cultures were amplified by passages on MS medium supplemented with 1.0 mg L−1 BAP + 0.1 mg L−1 IAA. Further, shoot amplification (29.2 shoots per vessel) was achieved by subculturing of in vitro regenerated shoot clump on MS medium + 0.5 mg L−1 BAP + 0.1 mg L−1 IAA. The micropropagated shoots were subsequently transferred for root formation on half-strength MS medium + 2.0 mg L−1 Indole-3 butyric acid (IBA) with 89% success rate. The in vitro-regenerated shoots were also rooted ex vitro with 34% success. These plantlets were hardened in the greenhouse and transferred to the field. The established protocol is suitable for true to type cloning of mature female plant of M. dioica.

High frequency plantlet regeneration from nodal segment culture of female Momordica dioica (Roxb.)

We report an efficient in vitro propagation method for Terminalia catappa using nodal segments of a 15-year-old mature tree. The nodal segments were cultured on MS medium supplemented with 6-benzyladenine (BA; 0.5–3.0 mg l−1) or Kinetin (Kn; 0.5–3.0 mg l−1) for bud breaking and multiple shoot induction. About 85% of the explant responded (2.8 ± 0.41 shoots per node with 2.7 ± 0.14 cm length) within 15 days of inoculation in Murashige and Skoog medium fortified with 2.0 mg l−1 of BA. Further shoot multiplication was achieved by repeated transfer of mother explants and subculturing of in vitro-produced shoots on medium supplemented with various concentrations of BA (0.25–1.5 mg l−1) or Kn (0.25–1.5 mg l−1) or on their combinations. Optimal number of shoots and shoot length were recorded on MS medium supplemented with 0.25 mg l−1 of BA and 0.25 mg l−1 of Kn. The multiplied shoots were used for ex vitro rooting after treatment for 4 min with indole-3-butyric acid (IBA; 50–500 mg l−1) or α-naphthalene acetic acid (NAA; 50–500 mg l−1). About 80% of the shoots treated with 200 mg l−1 of IBA produced ex vitro roots with an average of 2.8 roots per shoot. Nearly 75% of these plantlets could be acclimatized within 5 weeks and successfully established in the field. This is the first report on micropropagation of T. catappa, which can be applied for further genetic transformation assays and pharmaceutical purposes.

Micropropagation of mature Terminalia catappa (Indian Almond), a medicinally important forest tree

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