Efficient Regeneration of Tobacco (Nicotiana tabacum L.) Plantlets from Cotyledon, Hypocotyl and Leaf Explants: An Excellent Model Plant for Gene Function Analysis

Tobacco has been widely used as a model plant for stable and non-stable gene function analysis. Successful Agrobacterium-mediated transformation mainly depends on in vitro regeneration of tobacco plant. However, a reliable and standard regeneration protocol of tobacco using multiple explants is limited. In this study, we established a reliable and reproducible regeneration protocol of tobacco using three different explants i.e. cotyledon, hypocotyl and leaf. Preliminary, surface sterilized tobacco seeds were germinated on growth regulator free MS medium. Thereafter, in vitro germinated explants were inoculated into Murashige and Skoog [1] media supplemented with different combination and types of growth regulators for callus induction and subsequent regeneration of plantlets. It was revealed that, regeneration ability of explants is greatly influenced by type and nature of the explant. Among the three explants, higher callus induction (95%) was obtained in MS medium supplemented with 2.0 mg l-1 kinetin + 2.0 mg l-1 IAA from leaf explant. Also, leaf explant exhibited much higher regeneration ability (95%) than hypocotyl (60%) and cotyledon (45%) explants. Significantly highest number of shoots (8.0) were regenerated from leaf explants cultured on MS medium supplemented with 3.0 mg l-1 Kinetin+1.0 mg l-1 IAA compared to the other hormone combinations. Regenerated mature shoots were showed normal root after transferred onto ½ MS medium containing 0.3 mg l-1 IBA. This study will provide valuable information related to in vitro regeneration of tobacco plantlets using cotyledon, hypocotyl and leaf explants and will be used as a standard protocol for Agrobacterium-mediated transformation for gene function analysis.

Rapid In Vitro Multiplication of Non-Runnering Fragaria vesca Genotypes from Seedling Shoot Axillary Bud Explants

Fragaria vesca L. has become a model species for genomic studies relevant to important crop plant species in the Rosaceae family, but generating large numbers of plants from non-runner-producing genotypes is slow. To develop a protocol for the rapid generation of plants, leaf explants were compared to single axillary bud shoot explants, both from in vitro-grown Fragaria vesca seedlings, as sources of shoots for new plant production in response to benzyladenine (BA) or thidiazuron (TDZ) combined with indolebutyric acid (IBA) on Murashige and Skoog’s Basal Salt (MS) medium. BA at 2.0 and 4.0 mg L−1 and TDZ at 1.5 mg L−1 promoted the greatest number of shoots produced per shoot explant. There were no IBA effects or IBA interactions with BA or TDZ. Significant interactions between BA and IBA, but not TDZ and IBA, occurred in leaf explant callus formation and % explants with callus at 6 and 9 weeks of culture and on shoots per leaf explant at 9 weeks. TDZ treatments produced uniformly high levels of callus but low numbers of shoots. The treatment generating the most shoot production was BA at 4.0 mg L−1 plus IBA at 0.50 mg L−1. After 9 weeks of culture, leaf explants of the non-runner-producing genotype Baron Solemacher had generated 4.6 shoots per explant with the best treatment, while axillary bud explants had generated 30.8 shoots with the best treatment. Thus, in vitro culture of shoot axillary bud explants can generate high numbers of clonal shoots from a single seedling plant in vitro.

In vitro callus induction of gerbera from leaf explant

The experiment was designed to evaluate the effect of growth regulators on leaf explant of Gerbera for callus induction. Various kinds of plant growth regulators such as 6-Benzylaminopurine (BAP), α-Naphthalene acetic acid (NAA), 2, 4-Dichlorophenoxyacetic acid (2, 4-D), Indole-3-acetic acid (IAA) were used to initiate cultures. These were added to Murashige and Skoog medium in different combinations and concentrations. Leaf explants cultured on MS medium supplemented with BAP+ 2, 4-D+ IAA in T4 treatment & BAP+ 2,4-D in T5 treatment showed the best results for callus induction. On the other hand callus was induced early in the combination of BA+ 2,4-D + IAA hormone in T5, T9 & T8 treatment respectively. The rate of callus induction was very low in BA + NAA combinations but it was much earlier.

Callus Induction of Piper betle L. Var Nigra Using 2,4-Dichlorofenoxyacetic Acidand 6-Benzil Aminopurin

Piper betle L var Nigra (black betel) is a member of Piperaceae family which has potential as medicinal plant due to its secondary metabolites. Callus culture is one of the alternative methods to elevate production of secondary metabolites. This study was aimed to determine the effect of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzil aminopurine (BAP) towards callus induction and growth, also to determine the most optimal variation of 2,4-D and BAP concentration for callus induction of black betel leaf explant. This study was a laboratory experimental study with complete randomized design. Black betel leaf explant was planted in Murashige and Skoog (MS) medium supplemented with 2,4-D and BAP growth regulators at concentration of 0.0 mg/L, 0.5 mg/L, 1.0 mg/L, 1.5 mg/L, 2.0 mg/L respectively. Parameter recorded for callus induction and growth including callus induction time (days), percentage of explant forming callus, fresh weight, dry weight, color and texture. After callus planted for 8 weeks, analysis was performed statistically. Result showed that 2,4-D and BAP supplementation to medium affected the growth of black bete l leaf explants. Additional concentration of 0.5 mg/L 2,4-D and 1.0 mg/L BAP growth regulators showed the fastest response in callus formation, at 7.25 days. Growth regulators of 2,4-D 0.5 mg/L and BAP 2.0 mg/L concentration produced the highest fresh and dry weight, at 0.6802 g and 0.0670 g respectively. The best treatment was used as a basis to produce secondary metabolites.