High frequency organogenesis in hypocotyl, cotyledon, leaf and petiole explants of broccoli (Brassica oleracea L. var. italica), an important vegetable crop

Abstract

Broccoli (Brassica oleracea L. var. italica) is an important, nutritionally rich vegetable crop, but severely affected by environmental stresses, pests and diseases which cause massive yield and quality losses. Genetic manipulation is becoming an important method for broccoli improvement. In the present study, a reproducible and highly efficient protocol for obtaining organogenesis from hypocotyl, cotyledon, leaf and petiole explants of broccoli (Brassica oleracea L. var. italica cv. Solan green head) has been developed. Hypocotyl and cotyledon explants were used from 10 to 12 days old aseptically grown seedlings whereas leaf and petiole explants were excised from 18 to 20 days old green house grown seedlings and surface sterilized. These explants were cultured on shoot induction medium containing different concentration and combination of BAP and NAA. High efficiency shoot regeneration has been achieved in hypocotyl (83.33 %), cotyledon (90.11 %), leaf (62.96 %) and petiole (91.10 %) explants on MS medium supplemented with 3.5 mg/l BAP+ 0.019 mg/l NAA 2.5 mg/l BAP+0.5 mg/l NAA, 4.0 mg/l BAP+0.5 mg/l NAA and 4.5 mg/l BAP+0.019 mg/l NAA respectively. Petiole explants showed maximum shoot regeneration response as compared to other explants. MS medium supplemented with 0.10 mg/l NAA was found best for root regeneration (100 %) from in vitro developed shoots. The regenerated complete plantlets were transferred to the pots containing cocopeat and successfully acclimatized. This optimized regeneration protocol can be efficiently used for genetic transformation in broccoli. This is the first comparative report on multiple shoot induction using four different types of explants viz. hypocotyl, cotyledon, leaf and petiole.

Investigating the In Vitro Regeneration Potential of Commercial Cultivars of Brassica

“…Zhang and Bhalla [34] reported huge variations (0-96.7%) in seven Australian cultivars of Brassica napus. Similar genotype-dependent variations among different Brassica species have been reported in several studies [23,33,[35][36][37][38][39][40]43,[45][46][47]49].…”

“…Regeneration in Brassica has been reported from several tissues and organs including leaves, stem sections, petioles, roots, hypocotyls, cotyledons, immature zygotic embryos, protoplasts and cell suspension cultures [16,27,[33][34][35][36][37][38][39][40][41]. Out of these, cotyledons, hypocotyls and roots have been frequently used for genetic transformation [2,16,19,37,42,43].…”

“…The regeneration of these cultivars, including that of Aari canola, could be further improved upon by optimizing the plant regeneration conditions. It has been observed that varying the media components affects the shoot formation efficiencies significantly [33,[35][36][37]42,43,[45][46][47]. The fact that huge variations have been observed among different explants, a generalized recommendation cannot be made.…”

“…The explants were subjected to regeneration under three different regeneration protocols termed as Brassica Regeneration Protocols, BRP-I [23], BRP-II [53] and BRP-III [19]. A number of protocols for the in vitro regenerations of different Brassica genotypes have been reported in the literature, which are highly specialized for a certain genotype and are often complicated [1,3,16,28,29,[32][33][34][35][36][37]40,[42][43][44][45][46]48,[54][55][56][57][58][59][60]. Further, different explant types have been shown to respond differently to different hormonal combinations [44], which is further complicated by the fact that regeneration in Brassica is highly variable among different genotypes [42].…”

Effect of potent cytokinin thidiazuron on in vitro morphogenic potential of broccoli (Brassica oleracea L. var. italica), an important vegetable crop

“…In vitro plant regeneration studies in broccoli using several types of explants have been reported by various workers, viz., peduncle (Christey and Earle 1991), anther (Chang et al 1996), hypocotyl (Zhong and Li 1993;Puddephat et al 2001;Kim and Botella 2002;Ravanfar et al 2009;Huang et al 2011), leaf tissue (Robertson and Earle 1986;Cao and Earle 2003;Kumar and Srivastava 2015) and cotyledon (Qin et al 2006;Ravanfar et al 2011). Thidiazuron, (N 0 -phenyl-N 0 -1,2,3-thidiazol-5-ylurea, TDZ) is a phenyl urea that has gained importance as a potent plant growth regulator (PGR) for in vitro regeneration systems of various crops (Murthy et al 1995;Guo et al 2005;Ravanfar et al 2014).…”