Abstract
Chlorophytum borivilianum is a critically endangered plant well known for its medicinal properties for diabetes mellitus, diarrhea, arthritis, sterility, and erectile dysfunction, etc. Due to low viability and long dormancy of seeds, in vitro regeneration is required for large scale cultivation of this plant. In the present study, direct plant regeneration was optimized using flower stalk as explant. Nodal segments of flower stalk were sterilized and kept for direct regeneration on different combinations of BAP and KIN supplemented media. The highest, 15.27 ± 1.14 number of shoots were produced on medium containing BAP (2 mg/L) per nodal segment. The multiple shoot clumps regenerated from flower stalk were separated carefully and kept on rooting media. A maximum of 16.87 ± 1.53 roots per plant was observed in MS media having 0.5 mg/L of NAA. The rooted plantlets were shifted into the pot containing soilrite for hardening and acclimatization. The genetic stability of hardened plants was confirmed by start codon targeted, and inter simple sequence repeats molecular markers. All the 18 randomly selected plantlets showed similar genetic homogeneity to the mother plant. It is the first report on in vitro regeneration along with the genetic fidelity analysis of the regenerated plantlets from flower Stalk of C. borivilianum. As the standardized method of regeneration and mass multiplication is quite efficient and genetically stable, the protocol will be useful for the large-scale production of C. borivilianum to meet the market demand.
Highlights
Chlorophytum borivilianum is a critically endangered plant well known for its medicinal properties for diabetes mellitus, diarrhea, arthritis, sterility, and erectile dysfunction, etc
The explants inoculated on SIM2 and SIM6 media showed small greenish callus like structure at nodal region after 13 - 15 days of culture initiation, which further turned into multiple shoot clumps (Figure 1(d))
During subculture cycle these multiple shoot clumps were trimmed off from the flower stalk segment and divided into 4 - 5 sections for proper growth and multiplication of shoots (Figure 1(e) and Figure 1(f)). These shoots clumps were allowed to grow for 8 - 10 weeks on shoot induction media under continuous subculturing (Figure 1(g))
Summary
Dried roots are most commonly used in traditional medicine due to their aphrodisiac and natural tonic properties [4] It is grown using seeds and tubers. The long dormancy period of C. borivilianum seeds, poor seed germination, and low seed viability (11% - 24%) limits its natural regeneration methods [6] Due to these is limitations, in vitro culture based methods are in demand and have been successfully used for micropropagation of different species of Chlorophytum [7] [8]. Tissue culture which includes callus induction, micro-propagation and in vitro tuberization has been reported in different species of Chlorophytum such as C. comosum [9], C. borivilianum [10], C. amanienese [11] and C. arundianceum [8]. The major concern for in vitro regenerated plants is the incidence of somaclonal variations in the form of DNA polymorphism, chromosomal aberrations and sequence changes as the culture techniques, composition of nutrient media, amount and, combinations of growth hormones and growth conditions can cause somaclonal variations in micro propagated plants [16]
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