Stimulation Effect of AgNO3 and CoCl2 as Ethylene Inhibitors on in- Vitro Organogenesis of Sunflower (Helianthus annuus L.)

Abstract

To achieve an effective and reliable shoot organogenesis protocol in Helianthus annuus L.,
effect of ethylene inhibitors including AgNO3 and CoCl2 were investigated. Cotyledonary explants of two
cultivars including CMS19 and Progress were cultured in MS medium with 2.0 mg L-1 BAP and 1.0 mg
L-1 IAA supplemented with different concentrations of AgNO3 (0, 1.5 and 3 mg L-1) and CoCl2 (3 and 6
mg L-1). Statistical analysis of data showed a significant difference among cultivars and ethylene
inhibitors concentrations for organogenesis parameters including shoot regeneration and number of shoots
per explant. Addition of ethylene inhibitors to culture medium improved regeneration frequency and
number of shoots per explant. Shoot generation was improved with increasing concentrations of CoCl2
but in the case of AgNO3, increasing the concentration gave adverse results. Treatment of explants with
AgNO3 in low concentration (1.5 mg L-1) improved organogenesis in Helianthus annuus. The highest
shoot regeneration was obtained in media supplemented with 6 mg L-1 CoCl2 (R/E= 63% and S/RE= 3)
and 1.5 mg L-1 AgNO3 (R/E= 73% and S/RE= 2).This study suggests that ethylene inhibitors, particularly
CoCl2, could be used to improve the efficiency of in vitro shoot regeneration and plant transformation
protocols of sunflower.
Key words: Cotyledon, Silver nitrate

Keywords

• Cotyledon, Silver nitrate, Cobalt chloride, Helianthus annuus L., Sunflower, Plant regeneration

Ayçiçeği (Helianthus annuus L.)’nin in vitro Organogenezi Üzerine Etilen İnhibitörleri Olarak AgNO3 ve CoCl2’nin Uyarıcı Etkisi

Abstract

Ayçiçeğinde (Helianthus annuus L.) etkili ve güvenilir bir organogenez protokoluna ulaşmak için,
etilen inhibitörleri olarak AgNO3 ve CoCl2‘nin etkisi araştırılmıştır. CMS19 ve Progress çeşitlerinden
elde edilen kotiledon eksplantları 2.0 mg/L BAP ve 1.0 mg/L IAA içeren MS ortamına farklı
konsantrasyonlarda AgNO3 (0, 1.5 ve 3 mg/L) ve CoCl2(3 ve 6 mg/L) ilave edilerek kültüre alınmıştır.
Yapılan istatistik analizine göre; eksplant başına sürgün sayısı ve sürgün rejenerasyonunu içeren
organogenez parametrelerinde etilen inhibitörlerinin konsantrasyonu ve çeşitler arasında istatistiksel
olarak önemli farklılıklar bulunmuştur. Kültür ortamına etilen inhibitörlerinin eklenmesi, eksplant başına
sürgün sayısını ve rejenerasyon frekansını artırmıştır. Sürgün rejenerasyonu CoCl2 konsantrasyonun
artışıyla artmış, ancak AgNO3 konsantrasyonunun artışına bağlı olarak azalmıştır. Düşük konsantrasyonda
(1.5 mg/L) AgNO3 uygulaması ayçiçeğinde (Helianthus annuus) organogenez artırmıştır. En yüksek
sürgün rejenerasyonu ortama 6 mg/l CoCl2 (R/E=% 63 ve S/RE= 3) ve 1.5 mg/L AgNO3 (R/E=% 73 ve
S/RE= 2) eklendiğinde elde edilmiştir. Bu araştırmada etilen inhibitörlerinin, özellikle CoCl2’nın
ayçiçeğinde bitki transformasyon protokolu ve in vitro sürgün rejenerasyon etkinliğinin iyileştirilmesinde
kullanılabileceği önerilmiştir.

Keywords

• Kotiledon, Gümüş nitrat, Kobalt klorür, Helianthus annuus L., Ayçiçeği, Bitki rejenerasyonu

References

1. Alibert G, Aslane-Chanab JC, Burns M (1994). Sunflower tissue and cell cultures and their use in biotechnology. Plant Physiol. And Bioch. 32: 31-44
2. Bais HP, Sudha G, Suresh B,Ravishankar GA(2000). AgNO3 influences in vitro root formation in Decalepisha miltonii Wight and Arn. Curr. Sci. 79: 894-898.
3. Baker CM, Munoz-Fernandez N,Carter CD (1999).Improved shoot development and rooting from mature cotyledons of sunflower. Plant Cell, Tiss. and Org. Cult. 58: 39-49.
4. Beyer EM (1976a). A potent inhibitor of ethylene action in plants. Plant Physiol. 58 (3): 268-271.
5. Beyer EM (1976b). Silver ion: a potent anti-ethylene agent in cucumber and tomato. HortScience. 11 (3): 175-196.
6. Biddington NL (1992). The influence of ethylene in plant tissue culture. Plant Growth Regulation, 11 (2): 173-187.
7. Ceriani MF, Hopp HE, Hahne G, Escandon AS (1992). Cotyledons: an explant for routine regeneration of sunflower plants. Plant Cell Physiol. 33: 157-164.
8. Chae SC, Kim HH, Park SU (2012). Ethylene Inhibitors Enhance Shoot Organogenesis of Gloxinia (Sinningia speciosa). The Scientific World Journal.

9. Chatfield SP, Raizada MN (2008). Ethylene and shoot regeneration: hookless1 modulates de novo shoot organogenesis in Arabidopsis thaliana. Plant Cell Rep. 27: 655-666.
10. Chi GL, Pua EC (1989). Ethylene inhibitors enhanced de novo shoot regeneration from cotyledons of Brassica campastrisspp in vitro. Plant Sci. 64: 243-250.
11. Chi GL, Pua EC, Goh CJ (1991). Role of ethylene on de novo shoot regeneration from cotyledons of Brassica campastris spp Pekinensis (Lour) Olsson in vitro. Plant Physiol. 96 (1): 178-183.
12. Chraibi KMB, Latche A, Roustan JP, Fallot J(1991). Stimulation of shoot regeneration from cotyledons of Helianthus annuus by the ethylene inhibitors, silver and cobalt. Plant Cell Rep. 10 (4): 204- 207.
13. Giridhar P, Indu EP, Vijayaramu D, Ravishankar GA (2003). Effect of silver nitrate on in vitro shoot growth of Coffee. Trop. Sci. 43 (3): 144-146.
14. Huang X L, Li XJ, Li Y, Huang LZ (2001). The effect of AOA on ethylene and polyamine metabolism during early phases of somatic embryogenesis in Medicago sativa. Physiol Plant. 113: 424-429.
15. Jha AK, Dahleen LS, Suttle JC (2007). Ethylene influences green plant regeneration from barley callus. Plant Cell Rep. 26: 285-290.
16. Kumar P, Dimps RC, Goh CJ (1998). Influence of petiole and lamina on adventitious shoot initiation from lamina explants of Paulownia fortunei. Plant Cell Rep. 17: 886-890
17. Lai CC, Yeh SD, Yang JS (2000). Enhancement of Papaya axillary shoot proliferation in vitro by controlling ethylene. Bot. Bull. Acad. Sin. 41: 203-212.
18. Lau OL, Yang SF(1976). Inhibition of ethylene production by cobaltous ion. Plant Physiol. 58 (1): 114- 117.
19. Osman MG, KhalafallaMM (2010). Promotion of in vitro Shoot Formation From Shoot Tip of Tomato (LycopersIcon esculentum mill. cv. Omdurman) by Ethylene inhibitors. Int. J. Curr. Res. 4: 082- 086.
20. Park EH, Bae H, Park WT, Kim YB, Chae SC, Park SU (2012). Improved shoot organogenesis of gloxinia (Sinningia speciosa) using silver nitrate and putrescine treatment. Plant Omics. 5: 6-9.
21. Paterson KE, Everett NP (1985). Regeneration of Helianthus annuus in bred plants from callus. Plant Sci.42: 126-132.
22. Power CJ(1987). Organogenesis from Helianthus annuus inbreds and hybrids from the cotyledons of zygotic embryos. Am. J. Bot. 74: 497-503.
23. Pua EC, Chi GL (1993). De novo shoot morphogenesis and plant growth of mustard (Brassica juncea) in vitro in relation to ethylene. Physiol. Plant. 88 (3): 467-474.
24. Purnhauser L, Medgyesy P, Czako M, Dix PJ, Marton L (1987). Stimulation of shoot regeneration in Triticum aestivum and Nicotiana plumbaginifolia Viv tissue cultures using ethylene inhibitor AgNO3. Plant Cell Rep. 6 (1): 1-4.
25. Roustan J, Latche A, Fallot J (1989). Stimulation of Daucuscarota somatic embryogenesis by inhibitors of ethylene synthesis: Cobalt and nickel. Plant Cell Rep. 8 (3): 182-185.
26. Sarrafi A, Bolandi AR, Serieys H, Berville A,Alibert G, (1996). Analysis of cotyledon culture to measure genetic variability for organogenesis parameters in sunflower (Helianthus annuus L.). Plant Sci. 121: 213-219.
27. Sarrafi A, Kayyal H, Rachid Al-Chaarani G, Cantin F,Chaline AS, Durielle E(2000). Inheritance of organogenesis parameter in cotyledons of sunflower (Helianthus annuus L.). J. Genet. Breeding. 54: 227-231.
28. Sharma A, Kumar V, Giridhar P, Ravishankar GA (2008). Induction of in vitro flowering in Capsicum frutescens under the influence of silver nitrate and cobalt chloride and pollen transformation. Electron. J. Biotechnol. 11(2):84-89.
29. Songstad DD, Duncan DR, Widholm JM (1988). Effect of 1-aminocyclopropane-1carboxylic acid, silver nitrate and non- bornadiene on plant regeneration from maize callus cultures. Plant Cell Rep. 7 (4): 262-265.
30. Witrzens B, Scowcroft WR, Downes RW, Larkin PJ (1988). Tissue culture and plant regeneration from sunflower (Helianthus annuus) and interspecific hybrids (H tuberosus × H. annuus).Plant Cell, Tiss. and Org. Cult. 13: 61-76.
31. Yang SF, Hoffman NE (1984). Ethylene biosynthesis and its regulation in higher plants. Ann. Rev. Plant Physiol. 35: 155-189.