A basic requirement for the application of tissuecultures in crop impr - PDF document

A basic requirement for the application of tissuecultures in crop improvement is either to make use of ordevise an in vitroplant regeneration system. Callus caneasily be obtained from various parts of the sugar beet(Beta vulgarisL.) plant including hypocotyls, cotyledons,leaves, petioles, roots, flower stalks, anthers, embryosand seeds. Two groups of researchers reported ahormone-autonomous (habituated) callus which did notrequire plant growth regulators for growth andsubsequent regeneration (De Greef & Jacobs, 1979;Saunders & Shin, 1986). Formation of adventitiousshoots or roots from callus was achieved by severalresearchers although at low frequencies. Hooker andNabors (1977) and De Greef and Jacobs (1986), forexample, obtained one plantlet only from callus derivedTurk J Bot25 (2001) 25-33 Callus Development and Indirect Shoot Regeneration from SeedlingExplants of Sugar Beet (Beta vulgarisL.) Cultured In VitroSongŸl G†RELSugar Institute, Plant Breeding Department, Etimesgut, 06790 Ankara, TURKEYEkrem G†RELAbant Izzet Baysal University, Department of Biology, 14280 Bolu, TURKEYZeki KAYAMiddle East Technical University, Department of Biology, 06110 Ankara, TURKEYReceived: 23.02.2000Accepted: 28.09.2000Abstract:For callus production, hypocotyl, cotyledon, petiole and leaf explants from seedlings of different sugar beet (Beta vulgarisL.) breeding lines were cultured on MS medium containing BAP or KIN in combination with NAA or 2,4-D at 0.0, 0.5 or 1.0 mg/l.The use of both auxins in combination with 0.5 mg/l BAP or 0.5 mg/l KIN resulted in greater amounts of callus from all types ofexplants. However, hypocotyl and cotyledon explants produced significantly more callus than petiole and leaf explants when themeans of all lines were taken into account. Two types of callus were usually obtained, white and friable callus with large cells (TypeI) and green and compact callus with smaller cells (Type II). For shoot induction, Type I callus was transferred to MS mediumcontaining 2.5 mg/l TDZ or 1.0 mg/l BAP in combination with 0.3 mg/l IAA, together with or without a cold pre-treatment at 4¡Cfor two weeks. More shoots developed from the pre-treated callus and elongated shoots could be readily rooted on MS mediumcontaining 3.0 mg/l IBA.Key Words:Sugar beet, seedling explants, callus formation, indirect shoot regeneration. KŸltŸre AlÝnmÝß Þeker PancarÝ (Beta vulgaris L.) Fide EksplantlarÝndan Kallus Gelißimi veÜndirek SŸrgŸn Rejenerasyonu…zet :Kallus Ÿretimi iin, farklÝ ßeker pancarÝ (Beta vulgarisL.) Ýslah hatlarÝnÝn fidelerinden alÝnan hipokotil, kotiledon, yaprak sapÝve yaprak eksplantlarÝ, BAP veya KIN ile NAA veya 2,4-DÕnin 0.0, 0.5 veya 1.0 mg/l dŸzeyindeki kombinasyonlarÝnÝ ieren MSortamÝnda kŸltŸre alÝnmÝßlardÝr. Her iki oksinin de 0.5 mg/l BAP veya 0.5 mg/l KIN ile kombinasyonlarÝ, bŸtŸn eksplant tiplerindedaha fazla miktarda kallus olußturmuß fakat, bŸtŸn hatlarÝn ortalamalarÝ dikkate alÝndÝÛÝnda, hipokotil ve kotiledon eksplantlarÝyaprak sapÝ ve yaprak eksplantlarÝndan daha fazla kallus olußturmußtur. Genellikle, iki tip kallus elde edilmißtir; bŸyŸk hŸcrelerdenolußan beyaz ve gevßek kallus (Tip I) ve kŸŸk hŸcrelerden olußan yeßil ve sÝkÝ yapÝlÝ kallus (Tip II). SŸrgŸn Ÿretimi iin, Tip I kallusunbir kÝsmÝ karanlÝk ortamda 4 ¡CÕde iki haftalÝk bir soÛuk šn-ißlem uygulandÝktan sonra, diÛer bir kÝsmÝ ise šn-ißlem uygulanmadan2.5 mg/l TDZ veya 1.0 mg/l BAP ile 0.3 mg/l IAAÕnÝn kombinasyonunu ieren MS ortamÝna aktarÝlmÝßlardÝr. SoÛuk šn-ißlemiuygulanan kallusta daha fazla sayÝda sŸrgŸn elde edilmiß ve bŸyŸyen sŸrgŸnler 3.0 mg/l IBA ieren MS ortamÝnda kolaycaAnahtar SšzcŸkler:Þeker pancarÝ, fide eksplantlarÝ, kallus olußumu, indirek sŸrgŸn rejenerasyonu Research Article from young seedling explants. The composition of theculture medium is important in determining themorphogenetic pathway. A cytokinin (usually BAP) and anauxin (mostly IAA, NAA or 2,4-D) are normally includedin the primary culture medium for callus formationfollowed by incorporation of a lower auxin to cytokininratio for shoot induction and a higher ratio for rooting inthe subsequent media. Genotypic variation is alsoimportant in relation to callus production and subsequentorgan formation, some genotypes being more amenableto organogenesis than others and young tissues beingmore responsive than older tissues (Bhat, Ford-Llyod &Callow, 1985; Keimer, 1985; Mikami, Sudoh &Kinoshita, 1989; GŸrel, 1997). The present report describes an optimisedregeneration system in four sugar beet breeding lines,previously developed at the Sugar Institute (Ankara),from callus derived from different seedling explantscultured on a variety of medium compositions. Theregenerant plants will then be used in the further cyclesof our on-going breeding programmes.Materials and MethodsSeeds of four sugar beet breeding lines (Table 1) weretreated with 70% (v/v) alcohol for 5 min, and sterilisedwith 7.5% (v/v) sodium hypochloride for one hour in thepresence of 0.5 ml Tween 20 per 100 ml solution. Seedswere then rinsed several times with sterile distilled waterand left in sterile distilled water for 16-20 hours. Aftersterilisation, seeds were cultured on MS medium(Murashige & Skoog, 1962) containing 3% (w/v)sucrose, 0.8% (w/v) agar (Oxoid No. 3), 0.5 mg/l TIBAand 1.0 mg/l BAP. The pH was adjusted to 5.8 with 0.1M NaOH before adding agar and then autoclaving wascarried out for 15 min at 15 lb/sq in.For callus induction, hypocotyl and cotyledon explantswere taken from 10-12 day-old seedlings, and petioleand leaf explants were excised from 22-25 day-oldseedlings when they had 3-4 true leaves. Isolated explants(3-4 mm) were cultured on MS medium containing BAPor KIN in combination with NAA or 2,4-D at 0.0, 0.5 or1.0 mg/l each. All of the media combinations weresupplemented with 3% (w/v) sucrose and 0.8% (w/v)agar. Explants were cultured at 25±1¡C under a 16 hFor shoot regeneration, white and friable callusobtained from the above experiments was transferred toMS medium containing 2.5 mg/l TDZ alone or 0.3 mg/lBAP in combination with 0.1 mg/l IAA under darkconditions together with or without a cold pre-treatmentat 4 ¡C for two weeks. Callus was then transferred ontoa shoot regeneration medium containing either 2.5 mg/lTDZ alone or 1.0 mg/l BAP in combination with 0.3 mg/lIAA and cultures were incubated at 25±1¡C under a 16 hphotoperiod. The developed shoots were then rooted onMS medium containing 3.0 mg/l IBA only.Experiments were repeated three times, each with 10replicates, and callus and shoot formation were recordedafter 60 daysÕ incubation. The amount of callus wasestimated by a Ô0-4 ScaleÕ scoring system (Table 2) andthe number of shoots developed from the callus wascounted. Analysis of variance was carried out and thedifferences between the treatments were determined byDuncanÕs Multiple Range Test.AbbreviationsBAP: 6-benzylaminopurine, 2,4-D: 2,4-dichlorophenoxyacetic acid, GA3: gibberellic acid, IAA:indole-3-acetic acid, IBA: indole butyric acid, KIN: kinetin,NAA: naphtalene acetic acid, TDZ: thidiazuron, TIBA:2,3,5-triiodobenzoic acid.Callus Development and Indirect Shoot Regeneration from Seedling Explants of Sugar Beet (Beta vulgarisL.) Cultured In Vitro Table 1. Main characteristics of the sugar beet breeding lines usedLinesCharacteristicsM114Diploid monogerm, O-type, good root yield, medium M1017Diploid monogerm family, N-type beetELK345Diploid multigerm, good root yield, better sugar yield‚BM315Tetraploid multigerm, good root yield, good sugar yield Table 2.A Ò0-4 ScaleÓ scoring system developed for measuring theScoreDescription0No visible callus1Small proliferation at cut ends only25 mm callus at cut ends 35-10 mm callus from all over the explant4� 10 mm callus from all over the explant Callus Formation from Hypocotyl, Cotyledon,Petiole and Leaf ExplantsThe amount of callus developed from hypocotylexplants increased with increasing BAP concentrations inthe culture medium. The highest BAP concentration (1.0mg/l) resulted in significantly more callus production thanother BAP (0.1 and 0.5 mg/l) and KIN (0.1, 0.5 and 1.0mg/l) levels (Table 3). However, higher amounts of calluswere obtained when 0.5 or 1.0 mg/l 2,4-D was combinedwith a cytokinin, especially with 0.5 or 1.0 mg/l BAP.Combinations with 2,4-D in general producedsignificantly more callus than with NAA, with a mean of1.9-2.2 compared to 1.0-1.7, respectively.Cotyledon explants responded slightly differentlyfrom hypocotyl explants. 0.5 or 1.0 mg/l BAP producedsignificantly more callus than the control treatment and0.5 or 1.0 mg/l KIN (Table 4). The auxins also played animportant role in callus induction from cotyledon explantsand lower concentrations were found more effective. In the absence of cytokinins, petiole explants from alllines gave the lowest amount of callus (0.2) (Table 5).1.0 mg/l BAP resulted in the highest amount of callus(1.4), which was significantly different from the means ofthe control and other treatments. KIN also producedmore callus than the control treatment although it wasless effective than BAP. On the other hand, petioleexplants also produced significantly more callus in theS. G†REL, E. G†REL, Z. KAYA Table 3. Effects of different concentrations and combinations of auxins (NAA and 2,4-D) and cytokinins (BAP and KIN) on mean caRegulatorsBAP KIN (mg/l)0.00.51.00.51.0MeansNAA0.0 0.1 ± 0.10.8 ± 0.21.7 ± 0.10.7 ± 0.10.3 ± 0.11.0 d0.51.9 ± 0.21.7 ± 0.12.0 ± 0.21.5 ± 0.21.3 ± 0.21.6 c1.01.4 ± 0.22.0 ± 1.21.8 ± 0.21.9 ± 0.10.8 ± 0.21.7 c2,4-D 0.50.9 ± 0.22.3 ± 0.82.9 ± 0.11.4 ± 0.21.8 ± 0.21.9 b1.02.1 ± 0.21.9 ± 0.12.5 ± 0.22.3 ± 0.22.2 ± 0.22.2 a1.5 c1.8 b2.2 a1.6 bc1.3 d Table 4.Effects of different concentrations and combinations of auxins (NAA and 2,4-D) and cytokinins (BAP and KIN) on mean calRegulatorsBAP KIN (mg/l)0.00.51.00.51.0MeansNAA0.0 1.8 ± 0.22.0 ± 0.21.8 ± 0.21.1 ± 0.21.5 ± 0.21.6 bc0.52.0 ±0.22.0 ± 0.22.2 ± 0.21.6 ±0.21.5 ± 0.21.8 ab1.01.5 ± 0.21.8 ± 0.11.4 ± 0.11.8 ± 0.21.0 ± 0.21.5 c2,4-D 0.52.1 ± 0.31.8 ± 0.22.1 ± 0.11.5 ± 0.12.0 ± 0.21.9 a1.01.9 ± 0.71.8 ± 0.22.3 ± 0.11.8 ± 0.11.5 ± 0.21.7 abc1.7 bc1.9 a1.9 a1.6 c1.5 c either 0.5 mg/l BAP or KIN, or on medium containing no Table 5. Effects of different concentrations and combinations of auxins (NAA and 2.4-D) and cytokinins (BAP and KIN) on mean caRegulatorsBAP KIN (mg/l)0.00.51.00.51.0MeansNAA0.0 0.2 ± 0.10.7 ± 0.10.9 ± 0.10.3 ± 0.10.1 ± 0.10.4 c0.50.3 ± 0.10.2 ± 0.11.5 ± 0.21.2 ± 0.11.1 ± 0.20.9 b1.00.0 ± 0.01.3 ± 0.11.2 ± 0.20.4 ± 0.10.4 ± 0.10.8 b2,4-D 0.50.4 ± 0.12.3 ± 0.20.8 ± 0.21.1 ± 0.21.5 ± 0.21.2 a1.00.0 ± 0.01.4 ± 0.22.4 ± 0.21.5 ± 0.20.2 ± 0.11.2 a0.2 e1.1 b1.4 a0.9 c0.8 d Table 6.Effects of different concentrations and combinations of auxins (NAA and 2.4-D) and cytokinins (BAP and KIN) on mean calRegulatorsBAP KIN (mg/l)0.00.51.00.51.0MeansNAA0.0 0.4 ± 0.11.0 ± 0.20.9 ± 0.11.0 ± 0.20.8 ± 0.20.8 cd0.50.2 ± 0.10.3 ± 0.11.3 ± 0.20.6 ± 0.1-1.0 ± 0.10.7 d1.00.0 ± 0.01.3 ± 0.11.3 ± 0.10.8 ± 0.10.4 ± 0.10.8 c2,4-D 0.50.2 ± 0.12.0 ± 0.10.7 ± 0.11.2 ± 0.11.4 ± 0.11.1 b1.00.0 ± 0.01.7 ± 0.12.1 ± 0.11.6 ± 0.11.6 ± 0.21.4 a0.2 c1.2 a1.2 a1.1 b1.1 b S. G†REL, E. G†REL, Z. KAYA LinesEXPLANT TYPEHypocotylCotyledonPetioleLeafMeansM1142.0 a1.9 a0.5 d0.7 c1.3 abELK3451.3 b1.8 ab0.9 b1.2 b1.3 ab‚BM3150.6 c1.1 c0.7 c0.8 c0.8 bM10171.9 a1.6 b1.9 a1.5 a1.7 a1.5 a1.6 a1.0 b1.0 b Table 7. Effects of different explant types Figure 1.White and friable callus(Type I) derived from leafexplants of line M114 cul-tured on medium contain-ing 0.5 mg/l BAP only. Figure 2.Green and compact callus Shoot Regeneration from CallusThe development of shoots (Figure 3) from white andfriable callus (regardless of the explant type) wasobserved after 3-4 weeks of culture on mediumcontaining i) 1.0 mg/l BAP in combination with 0.3 mg/lIAA or ii) 2.5 mg/l TDZ alone. Slightly more shoots wereobtained from cold-treated explants of each medium butthe differences between the treatments as well as lineswere not significant at p= 0.5 (Table 8). Over 90% of theregenerated shoots could be readily rooted when culturedon medium containing 3.0 mg/l IBA only (Figure 4).DiscussionCallus DevelopmentCallus induction has been reported from almost alltypes of tissues of sugar beet on various mediaformulations, which usually contained a combination of acytokinin and an auxin (Saunders & Shin, 1986; GŸrel,1997; Van Geyt & Jacobs, 1985; Freytag et al., 1988;Saunders & Tsai, 1999). Catlin (1990) obtained callusand plantlets from cotyledons on medium containing 0.2mg/l BAP only. An auxin-dependent callus was initiatedfrom leaf pieces of sugar beet on medium supplementedwith 1.0 mg/l IAA and 0.1 mg/l KIN (Coumans et al.,1982). The most favourable media for callus inductioncontained a combination of 0.1 mg/l 2,4-D and 1.0 mg/lBAP or 2.2 mg/l TDZ alone (Roussy et al., 1996).Organogenic callus from hypocotyl explants of sugar beetwas initiated at concentrations of 0.3 mg/l BAP and 0.1mg/l NAA (Jacq et al., 1992). Hooker and Nabors (1977)recorded an interaction between the type andconcentrations of plant growth regulators, and explantsource, a combination of 2,4-D and BAP resulting in aCallus Development and Indirect Shoot Regeneration from Seedling Explants of Sugar Beet (Beta vulgarisL.) Cultured In Vitro With cold treatmentWithout cold treatmentBAP (1.0)BAP (1.0)LinesIAA (0.3)TDZ (2.5)IAA (0.3)TDZ (2.5)MeansM1140.6 ± 0.20.5 ± 0.20.3 ± 0.20.2 ± 0.20.4ELK3450.8 ± 0.20.6 ± 0.20.4 ± 0.20.4 ± 0.10.6‚BM3150.6 ± 0.20.5 ± 0.20.5 ± 0.20.5 ± 0.10.5M10170.5 ± 0.10.5 ± 0.10.4 ± 0.10.5 ± 0.10.50.60.50.40.4 Table 8.Effects of cold pre-treatment andshoots per explant) from white Figure 3.Shoots developed fromwhite and friable callus(Type I) on medium con-taining 2.5 mg/l TDZ onlyafter a two weeksÕ pre-cul-ture under cold and dark higher callus production than the combinations of IAA orNAA with KIN. Similarly, we found that 2,4-D was moreeffective for callus induction than NAA as a source ofauxin in all types of explants tested. Krens and Jamar(1989) reported that auxins were more effective whencombined with low BAP levels (0.05 mg/l). We observedotherwise but it should be noted that we did not use awide range of BAP levels. Among the cytokinins used inour experiments, BAP was clearly more effective thanKIN, especially when used at 1.0 mg/l in combination with0.5 or 1.0 mg/l 2,4-D. Normally, two types of callus were observed in ourexperiments. Basal medium or media containing 0.5 mg/lBAP and 0.5 or 1.0 mg/l KIN usually produced white andfriable callus (Type I) regardless of the presence ofauxins, while medium containing high concentrations ofBAP (1.0 mg/l) alone or in combinations with auxinsproduced green and compact callus (Type II). Theproduction of white and friable callus from various sugarbeet explants has also been reported by others (Saunders& Daub, 1984; Nakashima et al., 1988; Ritchie, Short &Davey, 1989; Konwar & Coutts, 1990; Shimamoto et al.,1993; Zhong, Smith & Thomas, 1993). Recent studieshave suggested that the organogenic potential is relatedto callus structure. White and friable callus consisting oflarge cells was able to produce roots and shoots(Saunders & Daub, 1984; Konwar & Coutts, 1990;Shimamoto et al., 1993), while green and compact calluswith small cells showed no organogenic capacity ( Ritchie,Short & Davey, 1989; Tetu, Sangwan & Sangwan-Norreel, 1987).Callus initiation was also influenced by genotypes. LineM1017 was the most productive, whereas line ‚BM315was the least. De Greef (1978) recorded a genotypicvariation in the callusing ability of leaf explants of sugarbeet, with some genotypes being more responsive thanothers. This may suggest that there is a considerablevariation in the distribution of the genes responsible forthis response among different populations of beet (Bhat,Ford-Llyod & Callow, 1985). Most of the explants of line‚BM315 turned a dark colour and showed no furthergrowth. This may be attributed to high levels of phenolicsubstances produced by this breeding line. Such awounding response, which inhibited callus growth, wasalso noticed by Harms, BaktÝr & Oertli (1983) in sugarbeet and garden beet.Variations also existed between different types ofexplants in their ability to form callus. In our experiments,cotyledon and hypocotyl explants produced considerablymore callus than petiole and leaf explants, indicating thatthe source of explant is an important factor indetermining the rate of success in such tissue cultureexperiments. This may also suggest that levels ofendogenous hormones or their sensitivity might varybetween organs.S. G†REL, E. G†REL, Z. KAYA Figure 4.A rooted shoot on medium Shoot Regeneration from Callus The process of shoot regeneration is generallysignalled by the appearance of green globular areas oncallus followed by the emergence of either leaf-likestructures or shoot bud initials. It is well known thatshoot formation from sugar beet is often induced whencallus is cultured on medium containing BAP (Shimamotoet al., 1993; Saunders, 1982), TDZ (Roussy et al.,1996), KIN and GA3(Coumans et al., 1982) or IBA andBAP (Freytag et al., 1988). On the other hand, Saunders(1982) obtained bud formation at low frequencies fromhabituated callus in the absence of growth regulators. Weobserved no significant difference between mediumcontaining a combination of 1.0 mg/l BAP and 0.3 mg/lIAA, and medium containing 2.5 mg/l TDZ alone,regardless of a cold pre-treatment at 4¡C for two weeks.However, cold pre-treatment slightly increased shootformation from callus in all breeding lines used, which isin agreement with the results of other researchers(Coumans et al., 1982). In contrast to our findings, DeGreef & Jacobs (1979) reported that pre-treatment ofcallus with cold for 3-9 weeks had no effect on shootformation from callus. Histological studies showed thatorganogenic callus was characterised by regions ofmeristematic cells located near the callus surface(Shimamoto et al., 1993; Abe et al., 1991) and most ofthese shoots were considered to be of organogenic origin,based on their rosette leaf patterns (Yu, 1989).Development of a reliable in vitroregenerationprotocol for sugar beet is difficult due to its highlyheterogenous nature. 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