In vitro propagation of Vanilla tahitensis moore and the role of plant growth promoting rhizobacteria on crop survival and growth in the field

Vanilla is one of the most expensive spices after saffron and cardamon. Vanilla propagation is through stem cutting but this method is slow to induce shoot. In vitro propagation technology is a technique for easy propagation on a large scale and is quick to produce large number of planting materials. In this study, Vanilla tahitensis Moore was used. Nodes from 5 months old seedlings of V. tahitensis Moore were used as explant and cultured in NDM + 0.2 activated charcoal. Explants were divided into two parts based on maturity, Explant 1 (1st to 4th node) and Explant 2 (5th node and below). Each part of the explant was sterilized in three treatments. The treatments were Sterilization 1 (60% Clorox®), Sterilization 2 (60% Clorox®, 40% Clorox®) and Sterilization 3 (60% Clorox®, 40% Clorox®, 20% Clorox®) at 15 minutes each. Parameters (% contamination-free, % survival, % shoot formation) were recorded after 30 days. Experiments on shoot regeneration (Experiment 1) and shoot multiplications (Experiment 2) were conducted using nodes from the in vitro derived shoot as explant. In Experiment 1, explant was cultured on NDM + 0.2% activated charcoal + Benzylaminopurine (BAP) (0 mg/L, 0.05 mg/L, 1 mg/L, 3 mg/L, 6 mg/L and 9 mg/L). Parameters were % shoot regeneration, days to shoot, the number of shoots per explant and shoot length, with all parameters recorded after 60 days of culture. In Experiment 2 hormone combination were used with BAP (0.5 and 1mg/L) + Naphtaleneacetic acid (NAA) (0, 0.5, 0.1, 0.2, 0.3mg/L). Data were recorded after 60 days including days to shoot development, number of shoots and shoot length. Thereafter, the new shoot was cut and cultured again in the new media with the same treatments. The number of shoots and shoot length were recorded after 60 days. For the experiment of rooting, nodes from the in vitro derived shoots were cultured in NDM (0.2% activated charcoal) +Indole-3- butyric acid (IBA) and NAA (0, 0.3, 1and 2 mg/L). Data on the number of roots and roots length were recorded after 60 days of culture. In the next experiment, PGPR was used as a treatment, and plantlets around 6 months old were transferred to an ex-vitro environment (field). Plantlets were transferred to polybags and placed under 50% shading for 24 hours. Thereafter, plants were inoculated with 5 mL of bacterial suspension (UPMB10, Pseudomonas sp., Bacillus pumilus, and Paenibacillus sp.) at a concentration of 108 cfu mL-1. Data were recorded at 80 days after transplanting for percentage of survival, length of vines, number of nodes, length of internodes, number of leaves, length of leaves, width of leaves, number of aerial roots, length of aerial roots, number of roots, length of roots and stress measurement (POD and CAT). In the sterilization experiment, the results showed no significant interaction between explant parts and treatment sterilization for all parameters, hence only single factor is discussed. The results of sterilization and the explant section for all parameters showed significant differences except for the shoots initiation. Sterilization protocol 3 and Explant 1 had the highest percentage for % contamination-free and survival. Subsequently, in Experiment 1 there were significant differences between BAP treatments on the number of days to shoot initiation and the length of the shoots. 1 mg/L BAP treatment was significantly quick in producing shoots and had the longest shoot length. Meanwhile, in experiment 2, the explant in the media produced only a single shoot and a combination of 1 mg/L BAP and 0.3 mg/L NAA produced the shoot in the shortest number of days. Meanwhile, explant in the media combination of 1 mg/L BAP and 0.2 mg/L NAA produced the longest shoot length. The result of the second culture shows that multiple shoot production is possible with the highest number of shoots obtained in the media combination of 1 mg/L BAP and 0.2 mg/L NAA. Result from the length of shoot showed that the explant in the media combination of 0.5 mg/L BAP and 0.3 mg/L NAA produced the longest shoot. For root experiments, the results of the number of roots showed no significant difference between the concentration of IBA and NAA. Meanwhile, root length measurements showed significant differences between concentrations of IBA and NAA. Media containing 1 mg/L of IBA produced the longest root compared to other treatments. Finally, field establishment of the seedling using PGPR, resulted in enhanced stem elongation, internode length, number of roots, root length, and POD activity with significant difference between treatments. Plants inoculated with UPMB10 had the longest internode while B. pumilus showed the highest results on the length of vine, the number of roots, root length, and POD activity. The results of the number of nodes, the number of leaves, the length of the leaf, the width of the leaf, the number of aerial roots, the number of roots, the root length, and the activity of the CAT showed no significant difference between the treatment.

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