No abstract

A study on the effect of various phytohormonal combinations on in vitro propagation of Cocoyam [Xanthosoma sagittifolium (L.) Schott] was conducted to develop an improved and efficient in vitro regeneration protocol for its mass multiplication. Histological analysis to understand the in vitro regeneration pattern and genetic fidelity assessment of regenerated plants were also carried out. Single shoots excised from in vitro established cultures of X. sagittifolium were used as explants. Among the 32 different phytohormonal combinations tested, indirect organogenesis with intervening callus phase was observed on majority of the media combinations. Meristematic clump formation was optimally achieved on all the tested media combinations with maximum 43.54 ± 0.51 shoot primordia on MS medium containing 0.2 mg/L BAP + 0.1 mg/L NAA followed by 36.44 ± 0.76 shoot primordia on MS medium having 2.5 mg/L TDZ. Micro-morphological analysis of different morphogenetic structures revealed that the regeneration of cocoyam is well executed via meristematic nodules, shoot primordia formation that may evolve in to proper shoots. Adventitious shoots (> 2 cm) were successfully (100.00 ± 0.00%) rooted on the half-strength MS medium containing IBA (0.05–1.0 mg/L) and IAA (0.05–0.5 mg/L). The number of roots ranged from 0.78 ± 0.31 on the control half-strength MS medium to 13.94 ± 0.46 on half-strength MS supplemented with 1.0 mg/L IBA. Considering somaclonal variations as a potential restriction to in vitro multiplication of plants, genetic stability was assessed using 40 ISSR primers. The PCR amplification profiles obtained from all the tested propagules (calli, meristematic clumps, regenerated plantlets) were similar to the mother plants indicating the homogeneity of the individuals raised through the regeneration protocol reported here.

Fenugreek (Trigonella foenum-graecum) is a leafy vegetable and spice crop, native to Indian subcontinent and Eastern Mediterranean region. Phytoplasma infection symptoms were observed in fenugreek at ICAR-National Bureau of Plant Genetic Resources Regional Station, Jodhpur and Agricultural Research Station Mandore Jodhpur, Rajasthan, India. The first appearance of phytoplasma suspected symptoms of little leaf was recorded after 50 days of sowing in the months of January 2022. The major symptoms recorded were virescence, phyllody, shoot proliferation, witches-broom, little leaf, yellowing and overall stunted growth in 146 germplasm accessions at NBPGR research farm, Jodhpur and one major commercially cultivated variety RMT 305 at Mandore Jodhpur. Ten samples from symptomatic and five samples from asymptomatic fenugreek plants were collected and processed for total DNA extraction using the Qiagen DNeasy plant mini kit (Germany). The extracted DNA was amplified using nested PCR assays with universal phytoplasma detection primers for 16S rRNA gene (P1/P7 and R16F2n/R16R2) and secA gene specific primers (SecAfor1/SecArev3 and SecAfor2/SecArev3) (Schneider et al. 1995; Gundersen and Lee 1996; Hodgetts et al. 2008). The amplicons of ∼1.25 kb with 16S rRNA and ∼480 bp with secA gene specific primers were amplified in all symptomatic fenugreek samples. In negative control (asymptomatic plants) no amplification was observed with either of gene specific primers in gel electrophoresis. PCR amplified products from the six selected positive samples (FPP-NBPGR-J-01 to FPP-NBPGR-J-04 and FPP-MND-01 to FPP-MND-02) of 16S rRNA and secA gene, were sequenced from both ends. Sequences were deposited in the NCBI GenBank with accession numbers ON756108-ON756113 for 16S rRNA gene sequences and ON745809 to ON745814 for secA gene sequences. BLAST analysis of 16S rRNA and secA sequences revealed 100% sequence identity among themselves and 99.95 to 100% sequence identity with the earlier reported phytoplasma strains of aster yellows group related phytoplasma strains (GenBank Acc. No. MN239504, MN080270) belonging to Ca. P. asteris (16SrI group). Further analyses of the 16S rRNA and secA gene-based phylogenetic tree and the iPhyClassifier-based virtual RFLP analysis of 16S rRNA gene study demonstrated that the phytoplasma associated with fenugreek phyllody belonged to 16Sr group I (‘Ca. P. asteris’) and subgroup B (GenBank accession AP006628), with similarity coefficient of 1.0. Earlier association of 16Sr-II-D subgroup (Ca. P. australasiae) with fenugreek as host was reported from Pakistan (Malik et al., 2020). To the best of our knowledge, this is the first report of a ‘Ca. P. asteris’, 16SrI-B subgroup related phytoplasma strain associated with fenugreek phyllody in the world. The 16SrI-B phytoplasma strain is a widely distributed strain associated with several agricultural and horticultural crops of India (Rao 2021). This is not only the first instance of fenugreek phyllody disease found in India, but also the first instance of fenugreek phyllody caused by 16SrI-B subgroup phytoplasma worldwide. This report has epidemiological significance and needs immediate attention, as fenugreek is one of the most common seed spice crop being grown all over India.