In vitro development and regeneration of microcorms in saffron ( Crocus sativus L )

Saffron (Crocus sativus L.) being triploid in nature is propagated by vegetative means through corms. The natural propagation rate of such plant species is relatively low; therefore an in vitro multiplication technique like micropropagation has been used as an alternative method of propagation for saffron. In the present investigation, apical bud explants were cultured on different nutrient media supplemented with various concentrations of plant growth regulators. Micro-corm formation was observed on all the media combinations. Maximum number (10) and weight (1.54 g) of microcorms developed were observed on MS media supplemented with 6-benzyl amino purine (BAP) (2 mg/L) + naphthalene acetic acid (NAA) (0.5 mg/L) + paclobutrazol (1.5 mg/L). Cultural conditions under light or in dark did not affect the corm formation and growth. Shoot and root regeneration was observed in the microcorms developed under in-vitro conditions. Maximum number of shoot (11.6) and length of shoots (11.4 cm) was also observed on MS media supplemented with NAA (21.6 μM) + BAP (22.2 μM). Maximum number of roots (11) and length of roots (11.4 cm) were obtained on G-5 media containing NAA (21.6 μM) + BAP (22.2 μM). The above observations will be useful as the base to make a possible road way for production of quality planting material in saffron.


INTRODUCTION
Crocus sativus L. (Iridaceae) is cultivated in few countries of the world for its highly valued stigmatic lobes.Saffron being triploid (2n = 3x = 24) is sterile and is propagated vegetatively through corms.A corm survives for only one season, producing up to ten "cormlets" that eventually give rise to new plants (Deo, 2003).Corm production is a rate limiting factor in saffron propagation as rate of generation of daughter corms under natural conditions is low (Chahota et al., 2003) which results in limited availability of propagating material for field cultivation.
Micropropagation is very good alternative for quality planting material/seed production and large scale multiplication of disease free saffron (Ascough et al., 2009).The successful tissue culture protocol was developed in saffron by several workers (Sharma et al., 2008, Mir et al., 2010).Microcorm production under invitro conditions shows promise with respect to rate of multiplication and number of microcorms produced in saffron (Darvishi et al., 2007).Regeneration has been described from corm-derived callus cultures via somatic *Corresponding author.E-mail: drmhkhan8@gmail.com.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License  embryogenesis, organogenesis and protoplasts (Demeter et al., 2010;Mir et al., 2010) but with low frequencies of normal plant formation.The present study was undertaken for efficient corm multiplication and in-vitro regeneration in saffron.

Explants
The present experiment has been carried out during 2010-2012 at Biotechnology Laboratory of Central Institute of Temperate Horticulture, Rangreth, Srinagar.Saffron corms were grown under controlled conditions in polyhouse of CITH, Srinagar Farm.The bulbs were harvested just before the onset of flowering and apical buds were removed and used for microcorm production and regeneration experiment.Explants were thoroughly washed under running tap water and sterilized by dipping in 70% ethanol for 3-4 min followed by surface sterilization with 0.1% HgCl 2 for 10 min and rinsed 5 times with sterile distilled water.
Cultures were maintained at 25 ± 1°C under 16/8 h (light/darkness) photoperiod with a light intensity of approximately 4000 lux.

Microcorm formation and regeneration
Cultures were sub-cultured and transferred to fresh media after every 4 weeks.Observations with respect to shoot length, number of shoots, root length, number of roots, number of corms and weight of corms were taken after every six weeks.This study was carried out as a factorial experiment based on completely randomized design (CRD) with 48 treatments in 5 replications.

Statistical analysis
Each treatment was replicated 5 times and observations in stages of development were recorded periodically.The data was analyzed by comparing means using one way analysis of variance (ANOVA) and the significance was determined by Duncan's Multiple Range Test using SAS (v 9.2).

Corm multiplication
Maximum number (10) and weight (1.54 g) of microcorms developed were observed on MS media supplemented with 2 mg/L BAP + 0.5 mg/L NAA + 1.5 mg/L paclobutrazol followed by 8.8 average number of microcorms with 1.34 g average weight on G-5 media supplemented with 2 mg/L BAP + 0.5 mg/L NAA + 1.5 mg/L paclobutrazol (Table 1, Figure 1).Cultural conditions under light or in dark did not affect the corm formation and growth.Our results showed further  improvement over the protocols developed earlier (Mir et al., 2010).In vitro micro-corm production of saffron has been obtained by culturing leaf segments and apical buds (Sharma et al., 2008), shoot explants (Milyaeva et al., 1995) and ovary explants (Raja et al., 2007).Microcorm formation from apical bud takes only eight months under in-vitro conditions as against 22 months under field conditions.

Shoot and root regeneration
Apical buds started sprouting within 10 days of incubation on culture media; however, only those cultured at appropriate concentrations of NAA and BA produce multiple shoots.Maximum number of shoot (11.6) and length of shoots (11.4 cm) was also observed on MS media supplemented with 21.6 µM NAA + 22.2 µM BAP (Table 2, Figure 2).In the present investigation, the results on shoot regeneration revealed that NAA and BA are essential for shoot regeneration of saffron (Crocus sativus L.).
Majourhay et al. ( 2007) investigated the ability of different cytokinins to induce shoot formation.Shoot development on corm explants was promoted by cytokinins while corm formation and growth was promoted by ethylene exposure (Plessner et al., 1990).Maximum number of roots (11) and length of roots (11.4 cm) were obtained on G-5 media containing NAA (21.6 µM) + BAP (22.2 µM).Our results on root multiplication and elongation are better than that reported earlier from corms (Sharma et al., 2008;Raja et al., 2007).The above observations will be useful as the base to make a possible road way for production of quality planting material in saffron.In-vitro regeneration in saffron was reported earlier by Mir et al. (2010) Our results on micro-corm production and multiplication promise to bridge the gap between land availability for saffron cultivation and availability of quality saffron planting material and regeneration protocol, with help in the development and rapid clonal propagation of novel saffron plant material.

Table 1 .
In-vitro corm multiplication in saffron from apical buds.

Medium BAP mg L -1 NAA mg L -1 Paclobutrazol mg L -1 Number of microcorms Weight of corm (g) MS
Means followed by the same letter within the columns are not significantly different (P= 0.05) using Duncan's multiple range test.

Table 2 .
In-vitro shoot and root regeneration in saffron from apical buds.

µM) BA (µM) Shoot length (cm) Number of shoots Root length (cm) Number of roots
Means followed by the same letter within the columns are not significantly different (P= 0.05) using Duncan's multiple range test.