Biotization using piriformospora indica for induction of in vitro floral primordia in saffron(crocus sativus L.)

Abstract

The study entitled "Biotization using Piriformospora indica for induction of in vitro floral primordia in saffron (Crocus sativus L.)" was conducted at the Department of Molecular Biology and Biotechnology, College of Agriculture, Vellayani, Thiruvananthapuram during 2022-23. The objective of the study was to evaluate the effect of biotization using Piriformospora indica for culture establishment and induction of in vitro floral primordia in saffron. The explants namely corms collected from farmer’s field, Jammu were used for the study. The pure cultures of P. indica were collected from the Department of Agricultural Microbiology, College of Agriculture, Vellayani. The effect of biotization of corms using P. indica was evaluated both in potting medium (Treatment 1) as well as in Murashige and Skoog (MS) medium (Treatment 2). Non-biotized corms were used as control (Treatment 3). Corms were planted in sterile potting medium (perlite and compost in 1:1 ratio) during June and kept in shade conditions for 3 months. However, no sprouting or root induction was noticed and later the corms got dried due to fungal contamination. Another strategy attempted was inducing in vitro rooting and further biotization. For this, the corms were kept at 16ºC during August-October. 100% sprouting was observed in 50 days of treatment and the sprouts thus induced in vivo were maintained at 18ºC for elongation. Further, 120-day-old elongated sprouts were cultured in basal MS medium for root induction. Healthy roots appeared in 3 days and in vitro rooted plants were transferred to potting media incorporated with P. indica mycelium. However, roots of all samples got dried in a week. For attempting biotization in vitro, full corms and 1cm3 cut corms were used for initiating sterile cultures. Sprouts were initiated within 15 days from 1cm3 cut corms cultured in MS medium supplemented with 1.5ppm BAP (6 benzylaminopurine). Further, the sprouts induced both in vitro and in vivo were subjected for elongation in basal MS medium. Significant increase in shoot length was noticed in sprouts induced in vivo (kept at 16ºC) compared to sprouts induced in vitro. Flower buds were induced from elongated sprouts by sub culturing in MS 81 medium and 50% sprouts that were induced in vivo, developed in vitro floral buds. However, no floral buds were observed in elongated sprouts induced in vitro. Biotization of in vitro cultures (Treatment 2) was carried out using P. indica agar discs and derivatives of P. indica. 130-day-old elongated sprouts having in vitro roots were inoculated with P. indica agar discs in MS medium for colonization. However, colonization was not observed under microscopic analysis. 165-day-old elongated sprouts were also treated with derivatives of P. indica and compared with control. Elongated sprouts treated with derivatives of P. indica exhibited a significant increase in plant height compared to control. These sprouts were further sub-cultured for a period of 7 weeks in MS medium for inducing multiple shoots. 165-day-old elongated plantlets treated for 80 days with derivatives of P. indica produced more number of multiple shoots per corm compared to control. Further, the multiple shoots developed were cultured in MS medium supplemented with 9% sucrose for inducing in vitro cormlets. Multiple shoots pre-treated with P. indica produced larger and more number of cormlets compared to control. To conclude, sterile potting media (perlite-compost) was found not suitable for the establishment and growth of corms under field conditions of Vellayani region. Storage of corms at 16ºC during dormant stage is found critical for in vitro growth, rooting and flowering in saffron. In vitro treatment using derivatives of P. indica in saffron showed positive responses with respect to plant height, number of multiple shoots, number, and size of in vitro cormlets. Hence, derivatives of P. indica can be used for better in vitro establishment of cultures, multiple shooting, and in vitro cormlet production in saffron.