Optimization of benzyl amino purines (BAP) concentration and medium type on the induction of banana shoots (Musa acuminata Colla.) cv. Barangan Merah under in vitro condition

The use of optimum concentration of BAP and the right medium type can support shoot induction on the explant of banana sucker cv.Barangan Merah. The Research was conducted at the Laboratory of Tissue Culture, Faculty of Agriculture, Universitas Syiah Kuala. This research used a completely randomized 3x3 factorial design. The first factor was Benzyl Amino Purines (BAP) concentration at three levels of concentration, i.e. control, 3 mg.L-1, and 6 mg.L-1. The second factor was Musrahige & Skoog (MS) medium type at three levels, i.e. solid, solid-liquid, and liquid. Results showed that the BAP treatment of 3 mg.L-1 had the biggest response to the number of open midribs compared to other BAP treatments. The type of solid medium tends to shoot induction better than other types of medium. The contamination that occurred was 13.9% of the 72 explants planted. The contamination was caused by Mucor and Aspergillus fungi. The bacteria causing the contamination were gram positive bacteria (coccus) and gram-negative bacteria (coccus and bacilli).

Effect of Light Conditions on In Vitro Adventitious Organogenesis of Cucumber Cultivars

The response on callus and shoot formation under different light incubation conditions was evaluated in cucumber (Cucumis sativus L.). Four-day-old cotyledon explants from the inbred line 'Wisconsin 2843' and the commercial cultivars 'Marketer' and 'Negrito' were employed. A four-week culture was conducted on MS-derived shoot induction medium containing 0.5 mg L-1 IAA and 2.5 mg L-1 BAP, under an 8-h dark/ 16-h light regime, or by a one- or two-week dark pre-incubation followed by the same photoperiod. Significant differences were obtained for the regeneration of shoots in all cultivars. The response in both frequency and number of shoots under continuous photoperiod was at least 3-6 fold higher than with dark pre-incubation. The highest genotypes response was obtained by 'Negrito' and 'Marketer' with identical values. All explants formed callus, and in two of the three cultivars, the response on callus extension was not significantly affected by incubation conditions. The results clearly show that shoot induction under continuous photoperiod regime was beneficial for adventitious shoot regeneration in cucumber.

Multiple shoot induction and regeneration of Vanilla borneensis Rolfe - a critically endangered orchid of Assam, India

In the present investigation, a micropropagation protocol has been developed for Vanilla borneensis Rolfe – a critically endangered orchid through multiple shoot regeneration. Through in vitro multiple shoot regeneration from both nodal and shoot tip explants, maximum (100%) shoot induction was observed. The minimum time required for shoot bud induction was observed from the shoot tip (5–7 days) on medium supplemented with BAP (4.44 mM) + KIN (2.32 mM) as compared to the nodal explants. Maximum multiple shoot regeneration was observed from nodal explants on the medium supplemented with BAP (4.44 mM) + TDZ (6.82 mM). However, maximum shoot length was observed on the medium supplemented with BAP (4.44 mM) + 15% CW and the number of nodes (5.27±0.33) per shoot after 90 days. Maximum (80-100%) of root initiation was observed in almost all the concentrations of NAA. The shortest time of root initiation was found on the medium supplemented with NAA (5.37 mM). Further, acclimatization period was found to be 15 days with 70% acclimatization while 60% of survivability was observed in the field condition. This efficient micropropagation method of V. borneensis could be successfully used for mass propagation as well as conservation of the critically endangered wild orchid.

In Vitro Adventitious Regeneration of Artemisia annua L. Influencing Artemisinin Metabolism

Artemisia annua L. is a herbaceous plant belonging to the Asteraceae family, known for producing, although at low levels, the sesquiterpene lactone artemisinin (AN), which is highly effective against malaria. In this study, an in vitro regeneration process of A. annua L. using ‘Artemis’ progeny was established and the potential of tissue culture for inducing new variability in terms of AN metabolism of in vitro regenerated plants was investigated. Among the plant growth regulators tested, the cytokinin 6-benzyladenine (BA) at 4.4 μM in combination with the auxin indole-butyric acid (IBA) at 0.35 μM yielded the greatest frequency of shoot induction. The optimal multiplication medium contained BA at 0.9 μM and naphthaleneacetic acid (NAA) at 0.05 μM. Regenerated plants (RPs), after transferring to the greenhouse and subsequently to the field, were analyzed during the growth cycle at different sampling times, showing a peak of AN content 20 days before blossom. Variability among different RPs and sampling times, in terms of AN and its precursors dihydroartemisinic acid (DHAA) and artemisinic acid (AA) was observed. This suggests that adventitious shoot induction could provide a useful strategy to induce variability influencing artemisinin metabolism as a consequence of in vitro manipulation.

Temporal Control of Morphogenic Factor Expression Determines Efficacy in Enhancing Regeneration

Background: Regeneration of fertile plants from tissue culture is a critical bottleneck in the application of new plant breeding technologies. Ectopic overexpression of morphogenic factors is a promising workaround for this hurdle. Methods: Conditional overexpression of WUS and ARF5Δ was used to study the effect of timing the overexpression of these morphogenic factors during shoot regeneration from root explants in Arabidopsis. In addition, their effect on auxin-signaling activation was examined by visualization and cytometric quantification of the DR5:GFP auxin-signaling reporter in roots and protoplasts, respectively. Results: The induced expression of both WUS and ARF5Δ led to an activation of auxin signaling in roots. Activation of auxin signaling by WUS and ARF5Δ was further quantified by transient transformation of protoplasts. Ectopic overexpression of both WUS and ARF5Δ enhanced regeneration efficiency, but only during the shoot-induction stage of regeneration and not during the callus-induction stage. Conclusions: The overexpression of WUS and ARF5Δ both lead to activation of auxin signaling. Expression during the shoot-induction stage is critical for the enhancement of shoot regeneration by WUS and ARF5Δ.

A Reliable Regeneration Method in Genome-Editable Bell Pepper ‘Dempsey’

A reliable regeneration technique is critical for the improvement of pepper traits in the genome editing era. Recently, we reported that peppers were successfully and specifically edited using CRISPR tools, CRISPR/Cas9 and CRISPR/Cas12a (LbCpf1). Although genome-editing tools can be applied to modify peppers at the cellular level, feasible pepper regeneration techniques have not been developed. Therefore, we studied a pepper regeneration protocol for Capsicum annuum L. ‘Dempsey’, a bell pepper species that has been proven to be genome-editable. Three explant types were used in this study, including the first leaves, cotyledons and hypocotyls of pepper seedlings. The shoot buds of the tested explants were produced using 8 mg/L 6-benzylaminopurine (BAP)- and 6 mg/L indole-3-acetic acid (IAA)-containing shoot induction medium (SIM). The first leaves of the ‘Dempsey’ seedlings showed an average shooting rate of 69.8%, whereas the hypocotyls and cotyledons had approximately 25.5% and 19.5% shooting rates, respectively. The regenerated ‘Dempsey’ plants exhibited no alterations in fruit and fertile seed phenotypes. Furthermore, the parent ‘Dempsey’ and progenies of the regenerants were cytogenetically stable with the same chromosome numbers (2n = 24). Therefore, this regeneration protocol enables the precise molecular breeding of ‘Dempsey’ peppers when coupled with CRISPR tools.

Efficient regeneration protocol for callus and shoot induction from recalcitrant Phaseolus vulgaris L. explants under optimum growth conditions

Callus is the most significant morphogenic response obtained in plant tissue culture studies. It can be used for micropropagation or to create transgenic lines. Phaseolus vulgaris L. (common bean) is one of the economically important crops with a great nutritional value. However, very little effort has been made to regenerate callus from P. vulgaris explants. Six explants were used namely root tip, leaves, plumule, radicle, cotyledon and embryo to develop a callus from P. vulgaris. The minimum days for callus induction was 10 days in plumule, radicle and embryo explants, while the maximum was 15 days in cotyledon explants with the callus induction percentage of 75%. The largest callus was found to be 2.77 gm in weight and 2.5 cm in diameter in MS medium. Medium with different concentrations of plant growth regulators (PGRs) showed different growth pattern in callus induction. Culture medium with 1.50 mg/l of BAP, 0.50 mg/l of 2, 4-D and 0.10 mg/l of NAA showed the best result in callus induction. Higher concentration of BAP (2.00 mg/l), along with 0.25 mg/l of 2, 4-D was ideal for shoot regeneration and maturation. Shoot induction medium along with 2.00 mg/l of NAA concentrations were found to be best for rooting. It was found that plumule and radicle favor callus induction, however, they are also potent for shoot and root induction. Knowledge gained in this study will be useful in developing a standard protocol for plant regeneration from P. vulgaris explants and will also be useful in creating transgenic line of P. vulgaris.

Efficient Protoplast Regeneration Protocol and CRISPR/Cas9-Mediated Editing of Glucosinolate Transporter (GTR) Genes in Rapeseed (Brassica napus L.)

Difficulty in protoplast regeneration is a major obstacle to apply the CRISPR/Cas9 gene editing technique effectively in research and breeding of rapeseed (Brassica napus L.). The present study describes for the first time a rapid and efficient protocol for the isolation, regeneration and transfection of protoplasts of rapeseed cv. Kumily, and its application in gene editing. Protoplasts isolated from leaves of 3–4 weeks old were cultured in MI and MII liquid media for cell wall formation and cell division, followed by subculture on shoot induction medium and shoot regeneration medium for shoot production. Different basal media, types and combinations of plant growth regulators, and protoplast culture duration on each type of media were investigated in relation to protoplast regeneration. The results showed that relatively high concentrations of NAA (0.5 mg l−1) and 2,4-D (0.5 mg l−1) in the MI medium were essential for protoplasts to form cell walls and maintain cell divisions, and thereafter auxin should be reduced for callus formation and shoot induction. For shoot regeneration, relatively high concentrations of cytokinin were required, and among all the combinations tested, 2.2 mg l−1 TDZ in combination with auxin 0.5 mg l−1 NAA gave the best result with up to 45% shoot regeneration. Our results also showed the duration of protoplast culture on different media was critical, as longer culture durations would significantly reduce the shoot regeneration frequency. In addition, we have optimized the transfection protocol for rapeseed. Using this optimized protocol, we have successfully edited the BnGTR genes controlling glucosinolate transport in rapeseed with a high mutation frequency.