~nt Cell Reports (1997) 17: 73-76 © Springer- Verlag 1997

C. H. S. Carvalho' N. Bohorova- P. N. BordalloL. L. Abreu . F. H. Valicente . W. Bressan . E. Paiva

Type II callus production and plant regeneration in tropical maize genotypes

VALI CENTEF.H.1997

Received: 5 October 1996 I Revision received: 21 April 1997 I Accepted: 9 May 1997

Abstract A total of 113 maize inbreds adapted to tropi-cal conditions were evaluated for their tissue culture re-sponse. Additionally, four media combinations of 15 or30 !J.M dicamba with or without 88 !J.M AgNOJ were usedto study the effect of dicamba and AgN03 on type II cal-lus production and plant regeneration from 42 of the in-bred lines. Inbreds 48, 389 and 1345 of the populationsBR 105, BR 112, and Cate te, respectively, showed a highcapacity for type II callus production and plant regenera-tion. The production of type II calli increased significantlywhen the concentration of dicamba was changed from15 to 30 !J.M and when AgNOj was added to the medium.A synergistic effect between 88 !J.M AgNOJ and 30 !J.M di-camba (CM-30Ag medium) was observed, leading to ad-ditional production of type 11 callus. Medium CM-30Agallowed the best tissue culture performance and plant re-generation capacity.

Key words Maize· Type II callus . Plant regeneration .Dicamba . Silver nitrate

Introduction

"víaize embryogenic callus can be classified as type I or- type 11(Armstrong and Green 1985). Type I callus is a com-

pact mass of cells that generate somatic embryos showingcomplex and organized structures. They are easily obtainedfrom immature embryos. Type II callus is friable. embryo-

Communicated by J. Widholm

C. H. S. Carvalho (18l) . P. N. Bordallo . L. L. AbreuF. H. Valicente . W. Bressan . E. PaivaEMBRAPA/CNPMS (Brazilian Agriculture Research CorporationlNational Maize and Sorghum Research Center). Caixa Postal 151,35701-970 Sete Lagoas-MG. Brazil

N. BohorovaCYMMIT (lnternational Maize and Wheat lmprovement Center).Londres 40, Apdo. Postal 6-641. Mexico 06600 DF. Mexico

genic, and maintains the ability to regenerate plants overtime. Due to its friable nature, type 11callus allows the es-tablishment of cell suspension and protoplast cultures andis preferred for some types of in vitro manipulation. Un-fortunately, type II callus tends to be initiated at a lowerfrequency than type I in embryos and has been obtainedfrom fewer genotypes.

Many maize genotypes adapted to ternperate regions areable to regenerating plants from tissue culture (Vasil et aI.1984: Duncan et aI. 1985: Tomes and Smith 1985; Hodgeset aI. 1986). On the other hand, fewer genotypes adaptedto tropical areas have shown the capacity to producetype 11 callus and plant regeneration from tissue culture(Prioli and Silva 1989: Bohorova et a!. 1995).

Many groups have demonstrated the effect of AgN03on the enhancement of type 11 callus production and thepromotion of maize regeneration (Vain et a!. 1989a, b;Songstad et aI. 1991. 1992). AgNOj has been shown toaffect ethylene action by competing for its binding site(Beyer 1976), thus promoting an increase in type 11callusand plant regeneration (Songstad et a!. 1991). The AgN03effect has been observed in some maize genotypes (Vainet al. 1989a, b; Songstad et a!. 1991). However. in most ofthe experiments, AgN03 was used with 2,4-dichlorophen-oxyacetic acid, while only Songstad et a!. (1991) tested theeffect of D medium (Duncan et a!. 1985), which containsdicamba (15 !J.M) combined with AgN03. on type II callusinduction. Dicamba has been reported to increase somaticembryogenesis in some Gramineae such as maize (Duncanet a!. 1985; Bohorova et a!. 1995), wheat (Hunsinger andSchauz 1987) and Dactvlis glomerata (Gray and Conger1985).

ln this report we present data on the potential of tropi-cal maize inbreds to produce type 11callus and regenerateplants by screening tropical maize genotypes from theNational Maize and Sorghum Research Center (CNPMS/EMBRAPA) in Brazil. A synergistic effect between di-camba and AgN03 on type II callus production and plantregeneration is also discussed.

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Materiais and methods

Altogether, 113 tropical maize inbreds were screened to identify gen-otypes capable of producing type II callus with a high potential forplant regeneration (Table I). For callus initiation and maintenance,N6 salts (Chu et a1.l975), 30 g/l sucrose, 100 mgll casamino acids,30 mM glycine, 15 11Mthiarnine, 7.5 11M nicotinic: acid, 7.5 11Mpyridoxine, 550 11Mmyo-inositol, 6 mM proline, 30 11Mdicamba(Velsicol, Chicago, Ill.) and 2.3 g/l gelrite (CM medi um) were used.Medium pH was adjusted to 5.8 before autoclaving.

Each genotype was cultured in six Petri dishes (I OOx 15 mm) with12 embryos per dish collected from three to five plants. lmmatureembryos used as explants were collected 12-15 days after pollina-tion (1.0 to 2.0 rnrn), but during the winter, some embryos werecollected up to 20 days after pollination. Cultures were maintainedin the dark at 26±loC and transferred to fresh medium every 15 or20 days for type I or rype Il callus, respectively.

After screening the genotypes for culturability, 42 inbred linesavailable at that time and which represent a sample of tropical gen-otypes currently used in our breeding program were used to studythe effect of dicamba and AgN03 on type Il callus production andplant regeneration. Callus induction was performed using CM me-dium with 15 or 30 11Mdicamba (CM-15 and CM-30 media), with orwithout 88 11MAgN03 (CM-15Ag and CM-30Ag). AgN03 wasautoclaved with the media. Twenty-one days after plating, the per-centage of type Il callus was determined and the cultures were elas-sified as embryogenic (presence of somatic embryos) or non-em-bryogenic (absence of somatic embryos). Slow-growing and darkcalli were discarded during culture. After 8 months of culture, thenumber of type Il calli was re-scored, and ali the calli were used toregenerate plants. Fifty days after transferring the callus to regener-ation medi um, the number of regenerated plants per gram of calluswas evaluated.

Plant regeneration was obtained using five pieces of callus, ap-proximately 50 mg each, per Petri dish with six Petri dishes per in-

bred. A medium containing MS inorganic salts (Murashige andSkoog 1962),20 gll sucrose. 30 mM glycine, 15 11Mthiamine. 7.5 11Mnicotinic acid. 7.5 11Mpyridoxine. 550 11Mmyo-inositol and 2.3 g/lgelrite (RM medium. Prioli and Silva 1989) was used. Regeneratedplantlets (3-5 em tall) were transferred to 200-ml flasks for furthergrowth. Only these plants were considered for analysis.

The data were analyzed by the MSTAT software and the mediawere compared by Duncan's Test. The percentage of callus produc-tion was analyzed after arcsine transformation.

Results and discussion

During the sereening phase for eallus induetion using 113inbreds (Table 1) in the CM medi um, two morphotypes offriable eallus were observed. lnbreds 48, 109, and 705 pro-dueed soft, friable ealli embedded in a mueilaginous sub-stanee with many somatie embryos. This type of eallus issimilar to that deseribed by Fransz and Sehel (1991) forthe genotype A 188. lnbreds 398, 389, 299, 340 and 366produeed a more friable, non-mueilaginous eallus. withfewer somatie embryos, similar to CAT 100-1, as reportedby Prioli and Silva (1989). For evaluation, both types ofealli were eonsidered as type lI. It is possible that the vari-ation among these ealli is due to differenees in their devel-opmental stage, in a way similar to that observed by Wel-ter et a!. (1995). Callus that produeed mueilaginous tis suetended to maintain this eharaeteristie over time.

Type I and type II eaJlus eould be maintained for24 months for many genotypes. ln general our results

Table 1 Genetic backgroundof the maize genotypes tested.Each inbred that formed at leastsome type Il callus is shown initalics. Values in parenthesesindicate the number of self-pollination at CNPMS (v open-pollinated variety)

Genetic origin lnbred

CMS O I (Mezcla Amarilla) - CNPMSCMS 02 - CNPMSCMS 03 - Amarillo Cristalino - CNPMSCMS 04 - CNPMSCMS 14 - Pool 25 CIMMYT (5) - CNPMSCMS 15 - Pool 26 CIMMYTCMS 16 - Pool 33 CIMMYTCMS 17 - Pool 34 CIMMYT

CMS 22 - Cate te ColombiaCMS 24 - Amarillo Subtropical - CNPMSCMS 28 - Tuxpefio Amarelo (8) - CNPMSCMS 33 - CNPMSCMS 35 - Pool 18 CIMMYT (5)CMS 50 - CNPMSCMS 51 - CNPMSCMS 52 - Experimental variety CNPMSCMS 107 - CNPMSBR 105 - Suwan (8) - CNPMS

BR 106 - Tuxpefio - CNPMSBR 111 - Pool 21 CIMMYT (8)BR 112 - Pool 22 CIMMYT (8)BR 451 (B1anco Dentado Tropical 2 - CIMMYT)CateteCatete Água LimpaCatete ESAAztecaTuxpanExperimental hybrid developed at CNPMSSweet com - CNPMS

1233. 1234, 1229, 12311234723.662,672,676,677,686,696098,711,712298, 299,013,057,010.l2Q. 151, I 82, l.8Q, 146102.094, 109, 116, 122084,132,077,082,085,088,130,135,137,125,127699.710.722,389,394,400703273.411,416072073,074432,43991,87,89lI05. 1106, 1109, 1096, 1097, 1099, IIII34.991,992I 154,ll1l, 1149, 1176,048,704,705,706,707,708. 709, 728016.022,019,023,018,724565,052.053,279,549,569377,340,369,366,389,398,336,352, 365BR451 (v)13451346, 134713441351. 13521215040484,485

.",..

Table 2 Effect of time after initiation on the regeneration capacityof callus obtained from the inbred tine 048 maintained on CM me-dium (I/lI indicates type I callus with small secrors of rype 11)

Callus age Callustype Number of regenerated(months) (I or 11) plants/g callus

I I1Il 1.538 li 24.9

13 11 28.625 11 0.9333 II 5.0135 11 7.09

showed that plant regeneration capacity was usually high-est in 4-month-old callus and, depending on the inbred, re-mained high until 15 months after plating. This observa-tion is probably due to the selection ofthe calli sectors withsomatic embryos during callus subculturing. which in-creased the proportion of embryogenic cells. Calli olderthan 25 months, in general, showed low plant regeneration,but some inbreds, such as 048 (Table 2), showed good plantregeneration up to 35 months after plating.

In addition to callus age, the capacity for plant regen-____eration from long-terrn callus cultures appears to be related

to other factors. ln many cases, it was observed that plantregeneration increased or decreased independently of cal-lus age, suggesting the influence of some seasonal factor.During callus subculture, the number of somatic embryosin the callus did not remain uniform, but varied with time(Table 2). It is possible that the initiation and/or prolifera-tion of embryogenic cells oscillates with time. resulting inalterations in plant regeneration capacity.

The populations BR 105, BR 112. and Catete showed agood type II callus production and plant regeneration re-sponse. Some inbreds of the Catete group were also uti-lized by Prioli and Silva (1989). confirming the potentialof this material for type II callus production. Although wetried to optimize the culture conditions for ali inbreds dur-ing the screening procedure, not ali inbreds were grownduring the same season. It is possible that other inbredsmay also respond better if tested in different environments.Inbreds 1345,389 and particularly the inbred 048 have al-ready been utilized in other studies in our laboratory show-

.g consistently good performance during callus produc-[,on and plant regeneration.

Among the 42 inbreds plated to study the effect of di-camba and AgN03 using the media described as CM-15,CM-30, CM-15Ag, and CM-30Ag, 26 (62%) ofthe inbredsproduced type II callus in at least one medium. A signifi-cant interaction (data not shown) between genotype andinduction medium was observed, showing that the geno-type culturability was dependent on the medium utilized.To compare the effect of the four media, another analysiswas carried out using only genotypes that had no me-diaxgenotype interaction, with the same ranking in the me-dia tested (Table 3). Type II callus production increasedsignificantly when the concentration of dicamba changedfrom 15 11M (CM-15) to 30 11M (CM-30) and with the ad-dition of AgN03 (CM-15Ag and CM-30Ag) to the media.

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Table 3 Effect of dicamba ( 15 and 30 11M) and AgN03 (88 11M) onthe percentage of type 11callus formed. Means within the same rowfollowed by the same letter do not differ significantly (P>0.05) byDuncans test. Five Petri dishes per inbred with 12 immatures em-bryos plated in each were used tnt not tested)

lnbred Media used

CM-15 CM-30 CM-15Ag CM-30Ag

366 1.39 6.25 3.03 14.81389 4.35 7.08 7.20 12.88048 0.0 4.17 5.56 8.332

1345 5.68 1.39 19.44 22.22703 0.0 8.33 0.0 9.72

Mean 2.28 b 5.30 b 7.49 b 13.59 a

052 1.67 nt 8.33 nt340 7.78 nt 15.28 nt366 1.39 nt 3.03 nt389 4.35 nt 7.20 nt

1154 0.0 nt 3.63 nt048 0.0 nt 5.56 nt704 12.50 nt 0.0 nt705 1.54 nt 0.0 nt706 3.67 nt 25.0 nt

1344 12.01 nt 18.94 nt1345 5.68 nt 19.44 nt073 1 ,~ nt 9.72 nt_._1

186 0.0 nt 10.0 nt1106 3.33 nt 18.33 nt1109 0.0 nt 9.03 nt1215 16.67 nt 36.11 ntMean 4.55 b nt I 1.85 a nt

565 0.0 3.33 nt nt340 7.78 0.0 nt nt366 1.39 6.25 nt nt389 4.35 7.08 nt nt

1154 0.0 2.27 nt nt1141 0.0 1.81 nt nt048 0.0 4.17 nt nt

1176 9.72 19.44 nt nt1344 12.01 20.77 nt nt1345 5.68 1.39 nt nt1346 36.57 41.67 nt nt073 2.27 4.58 nt nt150 1.39 5.0 nt nt298 0.0 1.39 nt nt703 0.0 8.33 nt nt

Mean 5.41 b 8.50 a nt nt

A synergistic effect between AgN03 and dicamba (30 11M)in the CM-30Ag medi um was observed, leading to addi-tional production of type II callus. Medium CM-30Ag al-lowed the highest percentage of inbreds with embryogenicand type II callus and the highest percentage of type II cal-lus in each inbred. The highest percentage of inbreds thatregenerated plants and the highest number of regeneratedplants/g callus in each inbred were also observed in theCM-30Ag medium. In addition, this medi um showed thelowest percentage of slow-growing and dark calli, as a re-sult of their greater longevity (Table 4).

Not ali callus c1assified as embryogenic producedplants, and some c1assified as non-embryogenic producedplants, showing that this classification does not accuratelypredict the regenerative capacity of callus.

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Table 4 Regeneration potential of calli of maize inbreds culturedfor 8 months in four different media. Maize inbreds used: 1233. 1234.565.052.016.022.377.340.369.366.389,723. 1154. 1141. 1149.

1176,048,704.705.706.1344,1345.1346.1347.1351. 1352,073,084. 132. 102, ISO, 15 L 182. 186. 273. 298. 699. 703. 1105, 1106,1109,1215

Medium lnbreds that lnbreds that Type II Slow-growing lnbreds that Mean numberproduced produced callus dark calli, regenerated of regeneratedtype Il embryogenic (%) 8 months after plants plants/gcallus (%) callus (%) planting (%) (o/c) callus

40.0 80.0 3.04 72.5 63.6 3.1266.6 85.7 5.79 66.7 57.1 3.1451.7 96.6 7.35 51.7 73.3 5.0680.0 100.0 13.02 40.0 83.3 7.67

CM-15CM-30DM-15AgCM-30Ag

Many calli that were classified as type II initially(2\ days after plating) were reclassified as type I after8 months. This probably occurred because callus showingat least one sector of type II callus at 21 days was classi-fied as such. Later observations showed that the mosteffective period ta isolate type II sectors was between 15and 25 days after embryo plating. Since the isolation ofsectors of type II was made at 50 days after plating, thetransition from type II to type I had already occurred.Changing from type II ta type I callus or vice versa canalso occur when the period of subculture is extended or thecomposition of the medium is changed (Vasil et a!. 1984:Mórocz et aI.1990).

This study has shown that many tropical maize geno-types can produce type II callus. Such information extendsthe range of genotypes that can be used in biotechniqueswhere tissue culture is involved, particularly when cell sus-pensions or protaplast cultures are required.

In our current protocol immature embryos \-2 mm insize are plated on CM-30Ag medium and cultured in thedark at 26°C. After 2-3 weeks, type II callus sectors areselected and subcultured separately every 2 weeks. AgNO,is usually added to the medium until calli are transferredta RM medium for plant regeneration. Alternatively,AgN03 can be removed after the second subculture. In RMmedium, plants are produced in approximately 30 days.

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