Korean Journal of Plant Resources (한국자원식물학회지)

The Plant Resources Society of Korea (한국자원식물학회)
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Optimization of Shoot Induction, Histological Study and Genetic Stability of in vitro Cultured Pisum sativum cv. 'Sparkle'

  • Kantayos, Vipada (School of Plant Production Science, Sunchon National University) ;
  • Bae, Chang-Hyu (Department of Well-being Resources, Sunchon National University)
  • Received : 2019.01.07
  • Accepted : 2019.02.10
  • Published : 2019.02.28
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Abstract

An efficient shoot regeneration condition for pea cv. 'Sparkle' was developed by using optimum explant, plant growth regulator concentrations, and pretreatment of BA onto explant. The average shoot number per explant showed the highest on two kinds of shoot induction media (MSB5 media containing 2 mg/L BA and a combination of 2 mg/L BA and 1 mg/L TDZ) when cotyledonary node explants were cultured. Moreover, the pretreatment of explant in 200 mg/L BA solution was found to be more effective in shoot induction than that of non-pretreatment. By histological study, cell division and proto-meristem were formed near the surface of the sub-epidermal and epidermal cell layers of cotyledonary node in earlier than 3 days after culture. The analysis of genetic stability of regenerants by using thirteen ISSR markers showed that in vitro regenerated plants showed polymorphism with 8.3% compared with their mother plants.

Keywords

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Fig. 1. Two kinds of explants used throughout this experiment. A: explant with cotyledonary nodes, B: half-split cotyledon originated from germinated Pisum sativum cv. ‘Sparkle’.

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Fig. 2. Shoot regeneration from half-split cotyledon explant of pea cv. ‘Sparkle’cultured on the basal MS + B5 vitamin and 2 ㎎/L BA combined with 1 ㎎/L TDZ after 45 days. A: above site, B: below site.

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Fig. 3. Shoot regeneration by pretreatment with BA onto explants in half-split cotyledon (A, B) and cotyledonary node explant (C, D). Explants were pretreated with 200 mg/L BA for 1 min on the basal MS with B5 vitamin and 2 ㎎/L BA (A, C) or not (B, D) in 21 days.

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Fig. 4. Number of shoots per explants by different types of explants (half-split cotyledon or coteledonary node) and pretreatment onto explants. Explants were pretreated with 200 ㎎/L BA for 1 min. on shoot induction solution containing BA 2 ㎎/L. †Within each sampling date, the results followed by the same letters are not significantly different according to DMRT (p ˂ 0.05).

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Fig. 5. Histological analysis of the shoot induction process in Pisum sativum cv. ‘Sparkle’. The cotyledonary nodes were cultured for 0 to 21 days on the basal MS with B5 vitamin and 2 ㎎/L BA. A: 3-day, B: 6-day, C: 12-day, D: 18-day. Abbreviations; ap: apical meristem, cn: cotyledonary node, ep: epidermal cells, h: hypocotyl area, L1: leaf primodia, m: meristem cells, vb: vascular bundle, vc: vascular cambium.

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Fig. 6. ISSR finger-prints generated using 13 primers from 30 accessions of Pisum sativum cv. ‘Sparkle’. Lane M: 100 bp DNA ladder marker, Lane M1-M3: mother plant, Lane1-10: regenerated plants. Arrows means positions of 4 polymorphic bands.

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Fig. 6. Continued.

Table 1. Shoots induction under different concentrations of plant growth regulators in 21-day

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Table 2. Thirteen ISSR primers and number of polymorphic bands amplified by each primers between regenerated plantlets and mother plants in pea cv. ‘Sparkle’

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