Thin Cell Layer Culture System: Regeneration and Transformation Applications [ Kiem Tran Kiem Tran Thanh Van (Editor), B. Van B. Van Le (Editor), Trevor A.
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Thin cell layer culture system [2003]
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This collusion ante is structure library to the model way treated by groups in SA Petite and Hisbiscus sabdariffa L. Other cytokinins like 2-iP can also induce adventitious bud induction in Bacopa either using individually or in combination with an auxin [21]. Thin cell layer TCL culture system is one of the options to ensure minimal use of tissue as an explant. By definition, thin cell layer culture is the use of small explants width: Depending on the incision made, an explant can either be longitudinal or transverse thin cell layer l or tTCL.
The effects of cytokinins, auxins, photoperiod, on successful regeneration and biolistic or bacterial-mediated transformation have been reported in species such as Nicotiana tabacum, Lilium longiflorum, Lycopersicon esculentum, Dendranthema x grandiflora, Eclipta alba, Orchids and various species of Rose thin cell layer cultures [23] - [28]. There are no reported studies that evaluate the effects of cytokinins on thin cell layer TCL explants of B.
Recent advances in metabolic engineering have made it possible to increase the concentration of desired compounds as well as introduce novel biosynthetic pathways leading into the production of novel bioactive compounds [29] [30].
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To improve metabolic engineering capabilities, it is important to have an efficient in vitro multiplication protocol in place. Genetic transformation studies that have been carried out in Bacopa genus so far have been summarized in Table 1. This study seeks to optimize adventitious shoot bud induction to facilitate herbal biomass production, role of liquid culture in shoot elongation, rooting and genetic transformation potential of tTCL explants.
A review of genetic transformation research on Bacopa monnieri. Bacopa plants that were growing in the greenhouse were sterilized using a procedure optimized in our lab [16]. Plant materials were harvested from the aseptic mother stock maintained on MS basal medium. The leaf and stem tTCL explants were aseptically excised using surgical blade No. Using fully expanded in vitro leaf and internode stem tissues, tTCL explants were prepared. The average initial tTCL leaf explant dimensions were less than 3 mm in length and 1 mm in width and the stem explant dimensions were about 1.
This medium was used to observe the rate of multiple shoot induction from using leaf and internode tTCLs explant by incorporating three concentrations of four cytokinins or compounds with cytokinins like activity. The pH was adjusted to 5. Five leaf and five stem tTCL explants were inoculated in each petri dish and three petri dishes for each treatment were inoculated.
After 14 days of incubation in the initial shoot induction medium with various cytokinins, explants with adventitious shoot buds were transferred to the elongation medium to induce further shoot growth. Plants were then removed from the mist chamber after a week and maintained in the greenhouse to monitor further growth. Samples were processed through fixation, dehydration, critical point drying and sputter coating prior to analyzing adventitious shoot bud induction under scanning electron microscope S N II, Hitachi High Technologies America Inc.
The protocol followed has been optimized in our lab and published earlier [32]. Coating was carried out for 60 s under a vacuum pressure of 0. Digital images were collected at various magnifications running beam at 10 kV and keeping specimen stub at 4. The standard protocol for Agrobacterium tumefaciens-mediated transformation of B. Dennis J Gray, University of Florida. Once the clean, isolated, and single conspicuous colonies were visible, they were picked to initiate new cultures. A single colony was selected to prepare bacterial culture for co-cultivation. Once the bacterial culture OD reaches between 0.
Explants were placed in the bacterial suspension for 25 min and the suspension was swirled occasionally to provide explant cut surface for attachment of Agrobacterium.
After 25 min of co-cultivation, explants were removed from the bacterial suspension and excess bacterial solution was removed by blotting explants on a sterile filter paper. The tTCL explants were placed in petri dish containing sterile filter paper and 3 mL of liquid MS medium was used to keep explants moist and maintain high humidity required for agro-infection. After washing explants for three hours in washing solution, cultures were transferred to semi-solid MS medium with 2. Transformation of 25 leaf and stem tTCL explants was repeated three times to score transformation frequency.
Tissue processing and extract preparation was carried out as described earlier [35].
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In brief, liquid nitrogen was used to grind 2 g of fresh and dried whole plant samples of B. The pellets in the tubes were further extracted two more times for 30 min each in 50 mL methanol. All extracted fractions were combined and passed through a 0. Rosemary Rosmarinus officinalis leaf extracts were used as the internal standard to compare antioxidant capacity. The absorption values were recorded at nm with Nanodrop c spectrophotometer Thermo Scientific, MA, USA for both assays after standard curves were generated.
The shoot induction experiments were carried out in triplicate for each treatment. A total of 15 explants from each type leaf and stem tTCL were used to score data. Once the optimum medium was identified, the same preparations were carried out in triplicate to record the shoot induction of the tTCL cultures. The experiments were set up in completely randomized design and repeated three times.
A one-way Analysis of Variance ANOVA between subjects was conducted to compare the effect of type of cytokinin and concentration on shoot induction for B. Employing visual observations like explant establishment, response time, explant necrosis and browning, and number of shoot buds generated during first four weeks of culture, MS medium proved more suitable. Subsequent experiment on adventitious shoot bud induction using tTCL explants were carried out by supplementing MS based medium with three concentrations of four cytokinins as outlined above.
Another preliminary study supported by qualitative observations revealed that the inclusion of auxin NAA even at 0. As our interest is multiple shoot induction, auxin NAA was omitted from shoot induction media treatments. Morphogenic response of leaf and stem tTCL explants has been shown in the Figure 1. Various stages in adventitious shoot bud induction of B. Various stages in elongation and rooting of B. Among all the cytokinins used, 2-iP exhibited poorest response as at 0.
Elongated shoots were removed from the elongation medium after three weeks and were photographed to record shoot length and root induction Figure 1 i. Other treatments like 0. The average numbers of shoots harvested from the most favorable treatment for leaf tTCL was 59 shoots per explant Figure 1 i and Figure 2.
To carry out liquid medium assisted elongation, culture assembly used is shown in the Figure 1 h. Bacopa shoots elongated rapidly and a clump of elongated shoot generating from tTCL explant looks like Figure 1 i. It is interesting to note that shoots elongated rapidly and some of the clumps that received lower levels of cytokinins concentration rooted as well Figure 1 j. Another notable feature of elongation phase was no incidence of hyperhydricity in Bacopa shoots. Majority of shoots rooted in this medium.
Notes Includes bibliographical references and indexes.
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