Euphytica 79 : 45-58, 1994 .© 1994 Kluwer Academic Publishers. Printed in the Netherlands .

Genetic analysis of in vitro plant tissue culture responses and regenerationcapacities

Y. Henry,', P. Vain' & J. De Buyser'1 Laboratoire de Biologie Moleculaire Vegetale, bdtiment 630, URA CNRS 1128, Universite Paris XI, 91405Orsay, France; 2 Laboratoire de Physiologie Moleculaire Vegetale, b6timent 630, URA CNRS 1128, UniversiteParis XI, 91405 Orsay, France

Received 19 July 1994 ; accepted 20 July 1994

Key words: genetics, tissue culture, regeneration, cytoplasm, embryogenesis

Summary

The in vitro development of a whole plant from a single cell (eg microspore or somatic cells) is a characteristicfeature of plants. The amenability of a plant to in vitro culture is influenced by the genotype, which is thus ofmajor importance in the plant tissue culture response . The differences observed between different cultivars duringin vitro tissue culture with respect to embryogenesis and regeneration result from quantitative or qualitative geneticdifferences. We describe results obtained from quantitative genetic studies, from Mendelian genetic analysis andfrom gene mapping . It is less easy to study the influence of cytoplasmic genomes . Moreover, it is necessary todiscriminate between maternal effects and cytoplasmic inheritance . A conclusion from this review is that the choiceof parental strains for a breeding program should be realized on agronomic criteria rather than on compatibilitywith the tissue culture technique used. Fortunately, it is relatively easy to incorporate short-term tissue culturecapacity into agronomically valuable genotypes . This is of major interest since tissue culture remains necessaryfor most aspects of crop plants biotechnology . Very little is known about the molecular events that trigger in vitroembryogenesis and regeneration . It is clear that genes involved in the tissue culture response are not specialised`tissue culture genes' .

Introduction

The capacity of gametophytic or sporophytic cells toform in vitro embryos which are competent to devel-op (i .e. to regenerate) is a particular feature of plants .In this regard, microspore and somatic embryogenesiscan be regarded as model systems to investigate themechanisms of plant embryogenesis and development,and the whole process of plant cell differentiation .The fact that embryos can develop from microsporeor somatic cells also demonstrates that the genetic pro-gram for embryogenesis can be completed outside ofsexual reproduction .

The levels of embryo induction and plant regener-ation from in vitro tissue cultures are basically influ-enced by the genotype and the physiological status ofthe donor plant, the plant organ used as an explant,the culture medium and the interactions between them

45

(Mathias & Simpson, 1986c ; Lazar et al., 1984a; Bre-gitzer, 1992). Despite considerable efforts, the regener-ation in vitro of cereal and legume species has remainedmostly empirical . Since the first successful experi-ments on gametic (Guha & Maheshwari, 1964) andsomatic (Stewards et al ., 1958) tissue culture, differ-ences between genotypes for in vitro response havebeen observed. It has become increasingly evident thatthe genotype used is of major importance for most planttissue cultures, including model species such as tobac-co and Arabidopsis (Masson & Paszkowski, 1992) .

It is trivial to observe differences between geno-types for in vitro responses :- More than 350 lines of maize are susceptible, atvarious levels, to regenerate plants through somaticembryogenesis from compact type I callus (Lu et al .,1982). However, only a few maize genotypes, fol-lowing the discovery of the inbred line A188 (Green,

46

1982), is able to do so from friable embryogenic typeII callus. These type II calli are susceptible both toproduce cell suspensions and to regenerate plants fromlong-term experiments and therefore were at the cross-road of many genetic manipulations of maize . 'Short-and long-term somatic embryogenesis were controlleddifferently in maize' (Tomes & Smith, 1985). Compa-rably for maize anther culture capacity: `The screen-ing of a wide range of genotypes for their response toanther culturing revealed a relatively small proportionof genotypes that were responsive' (Wan et al ., 1992) .- Nearly all wheat genotypes tested so far in vitrohave the potential to produce very short-term andro-genetic (Baroncelli et al., 1978 ; Ozias-Akins & Vasil,1982; Henry & De Buyser, 1990) and somatic embryo-genesis . However, wheat genotypes are quantitative-ly different for those traits, using up to date sys-tems of evaluation. For wheat anther culture (short-term culture), all the genotypes were more or lessresponsive to tissue culture, but large quantitative vari-ations between lines were clearly exhibited for fre-quencies of embryo induction, for embryo regenera-tion and for albinos/green plant regeneration ratio (DeBuyser & Henry, 1979). Comparably to maize, long-term wheat somatic embryogenesis and regenerationare also restricted to a few genotypes (Vasil, 1987 ; DeBuyser et al ., 1992a; Henry et al ., in press) .

These significant genotype effects suggest thatgenetic factors are primordial in the determination ofin vitro tissue culture response level . Therefore, thefirst step to involve in vitro culture in crop improve-ment is to identify plant genotypes that are compe-tent for in vitro culture. The genotype screening is along and expensive process . To date, even under thebest tissue culture conditions currently used, agronom-ically elite genotypes remain difficult to manipulatein vitro . Tissue culture performances are continuous-ly improved through non genetic factors affecting tis-sue culture response (Ouyang et al ., 1983 ; Masson &Pazkowsky, 1992). However, extensive culture condi-tions screening is often insufficient to overcome geno-typic dependency (Mathias & Simpson, 1986a) . Thedifferences observed between varieties for tissue cul-ture competence are still a limiting factor for breedingpurposes and to a lesser extent to more fundamentalresearch concerning plant development . The ability toregenerate in vitro-grown plant tissue remains a pre-requisite for their genetic transformation . The studyof tissue culture ability inheritance is also made diffi-cult by the large amount of uncontrolled and environ-mentally induced variation (Ockendon & Sutherland,

1987). Experimental designs allowing the separationof genetic and non genetic components are requiredfor such study (Ghaemi et al ., 1993) .

The objective of this paper is to summarise litera-ture concerning the genetic component of tissue cul-ture ability . We have focused on results obtained fromquantitative genetic studies, from mendelian geneticanalyses and from gene location . Additional sectionsare concerned with the influence of cytoplasm, and theconsequences of tissue culture competence variation inplant breeding .

Influence of the nuclear genome

Numerous experiments have shown that nuclear genescontrol the in vitro response. Results also suggest thatthe differences observed for plant tissue culture abilityresult from quantitative (mostly in short-term cultures)or qualitative (long-term experiments) genetic differ-ences. This distinction between short and long-termtissue culture competence is particularly important formonocotyledonous species .

a) Quantitative genetic studies

Diallel analysis of shoot formation from cauliflowertissue culture was performed as early as 1974 (Buiattiet al., 1974), indicating that gene action was additive,but with a low heritability . Additive gene action alsoaccounted for a large part of the shoot-forming vari-ation between tomato F1 s in a diallel cross (Franken-berger et al ., 1981) . Somatic embryogenesis in wheatappears to be influenced by additive, dominant andmaternal genetic effects . Ou et al . (1989) indicated thatmore than 80% of the observed genotypic variation forsomatic embryogenesis induction was due to additiveeffects . For maize somatic embryogenesis, additivevariance is high compared to non-additive or domi-nance variation (Nesticky et al ., 1983) . Additive genet-ic variance is also the most significant source of vari-ation for somatic embryogenesis in red clover (Keyeset al ., 1980), black spruce (Cheliak & Klimaszewska,1991), white spruce (Park et al ., 1993), rice (Peng &Hodges, 1989) and wheat (Chevrier et al ., 1990) . Mostof these results suggested that parents producing highlyresponsive hybrids can be identified . Genetic variationfor maize callus growth and shoot regeneration abilitywere observed in diallel analysis, although a large pro-portion of the variation was of the additive type, pos-itive heterosis increased the tissue culture response in

maize somatic embryogenesis (Beckert & Qing, 1984 ;Tomes & Smith, 1985 ; Willman et al ., 1989) . Evenmany differences between genotypes for in vitro tis-sue culture responses were reported to be additive andheritable (Keyes et al ., 1980 ; Tomes & Smith, 1985),some `genetic studies indicated that, compared to typeI, type II cultures have a higher proportion of non addi-tive genotype variation' (Tomes & Smith, 1985) . Onthe contrary, immature panicle culture of rice (Chu& Croughan, 1990) indicated dominance, additive xadditive and dominance x additive gene effects, withadditive effects of minor importance .

Studies designated to analyse the inheritance ofanther culturability in maize demonstrated that generalcombining abilities were highly significant (Petolino& Thompson, 1987), with a high heritability. A pre-dominantly additive gene action exists for microspore-derived embryo induction (Charmet & Bernard, 1984 ;Miah et al ., 1985 ; Deaton et al ., 1987; Wu & Chen,1987; Powell, 1988; Agache et al ., 1988; Quimio &Zapata, 1990; Koba et al ., 1993) and green plant regen-eration (Tuvesson et al ., 1989 ; Zhou & Konzak, 1992 ;Shimada et al ., 1993). Estimated heritabilities werehigh for anther culture ability (Charmet & Bernard,1984; Lazar et al., 1984b; Miah et al ., 1985) . Fortunate-ly, even with environmental variation being present,high heritability make androgenesis an easy trait tomanipulate (Becraft & Taylor, 1992) . This also allowsmendelian segregation ratios to be studied in manycases .

b) Mendelian genetic studies

Genotypes which produce callus, somatic or androge-netic embryos in vitro have been tested for their abilityto sexually transmit these traits, and sometimes recip-rocal crosses have been performed. All the analysesof segregating populations unequivocally demonstrat-ed that there is a genetic component for tissue-culturetraits . In wheat anther culture, green plant regenerationtrait segregated among backcross populations (Zhou& Konzak, 1992), and this trait was thus attributed tonuclear genes . In other instances doubled haploids pro-duced from F, hybrids were used for segregation stud-ies. In wheat, even immature embryo culture of recip-rocal Fi hybrids between embryogenic and nonem-bryogenic lines did not show somatic embryogenesis,F2 embryos segregated for this character, indicating anuclear genetic control of in vitro somatic embryoge-nesis (De Buyser et al ., 1992a). Anther culture exper-iments with petunia (Raquin, 1982), barley (Foroughi

47

et al ., 1982; Knudsen et al ., 1989), rice (Tsay et al .,1982) and wheat (Lazar et al ., 1984b ; Henry et al .,1984; Henry & De Buyser, 1985) have demonstrat-ed that embryogenesis (embryo rate), plant regener-ation, and for cereals, development of albino plantswere genetically controlled . Each of the three parame-ters was under separate genetic control . Similarly, formaize and potato, embryo production and plant regen-eration ability from anther culture were not correlated(Dieu & Beckert, 1986 ; Meyer et al ., 1993), indicatingalso that these traits `depend on different types of geneaction' . Similar evidence was obtained from somaticembryogenesis experiments, and it has been demon-strated that callus induction rate and regeneration abil-ity are controlled by independent genetic systems inwheat (Chowdhury et al ., 1991) and barley (Komatsu-da et al ., 1989) .

The lack of a significant correlation between theperformance for anther and somatic tissue cultures wasalso demonstrated, indicating separate genetic con-trol (Agache et al ., 1988) . Genes that control somaticembryogenesis appear to be independent of those thatcontrol anther culture (i .e . microspore embryogene-sis) in wheat (Agache et al ., 1988), rye (Lazar et al .,1987) and maize (Armstrong et al ., 1992) . Taylor &Veilleux (1992) also found no significant correlationamong anther culture competence, protoplast cultureability and leaf disc regeneration forSolanum phureja,implying three separate genetic mechanisms .

When anther culture or somatic tissue culture abil-ities were each considered as a single character, theinheritance was generally defined as complex (For-oughi et al ., 1982; Close & Gallager, 1989) or extreme-ly complex (Aslam et al ., 1990) . Most studies ded-icated to determine the number of genes controllingthe various components of tissue culture respons-es indicated that 1, 2 or a few nuclear genes wereinvolved (Table 1). Recessive nuclear genes controlledboth anther culture capacity in maize (Cowen et al .,1992), potato (Wenzel & Uhrig, 1981 ; Sonnino etal., 1989), rice (Miah et al ., 1985 ; Quimio & Zapa-ta, 1990) and somatic tissue culture capacity in wheat(De Buyser et al., 1992a) and Solanum phureja (Taylor& Veilleux, 1992) . Recessive genes were also respon-sible for regeneration capacity either from leaf disc intomato (Frankenberger et al ., 1981), or from immatureinflorescence callus in rye (Rakocszy-Trojanowska &Malepszy, 1993) . On the contrary for alfalfa (Reisch& Bingham, 1980; Wan et al ., 1988 ; Ray & Bingham,1989; Hernandez & Christie, 1990 ; Kielly & Bowley,1992; Yu & Pauls, 1993), Asparagus species (Delbreil

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Table 1 . Genetics of plant tissue culture

Ref # : literature reference number; Htc : haploid tissue culture (AC: anther culture) ; Stc : somatic tissueculture (SE: somatic embryogenesis, tI = type I, tII = type II ; R : plant regeneration, from : c : = callus, l =leaves and Id = leaf disc) ; P: protoplast culture; 0 : other tissue culture (pe = petiole, Id = leaf discs) .

Species Ref #

Htc Stc Gene numberAC SE R P 0

Alfalfa 120 rc 2 dominant142 + pe 2 complementary dominant (both

118 + penecessary for regeneration)few dominant

60

+ I dominant for callus production

68

+2 complementary for somatic embryogenesis2 dominant

148

+ 2 dominant complementaryAsparagus species 34

+ I dominantBarley 44

+ complexBrassica species 6

+ extremely complexMaize 17

tI dominant62

tI dominance for the formation of embryos123

tI at least 2 partially dominant7, 1

+ relatively simple27

? complex145

ti at least 114

+ major genes29

+ 2 major recessifs epistatics and 2 minors4

tlI + I major geneCotton 48

+ more than one geneCucumber 98 + 2 complementary dominantDacrylis glomerata 47

+ I dominantPetunia 63 + few

117

+ not monogenic38 pe 2 without dominance

Potato 144,130

+ more than one recessive129

+ I dominant for anther culture competence28 rl

+ relatively simple genetic controlRice 95

+ I recessive for callus induction frequency115

+ few recessiveRye 116 rc at least 2 recessive complementary lociSolanum 140

+ Id 2 for induction and 2 for regenerationchacoenseSolanum 23 + 2 dominantphureja 133

+ I dominant133 + 2 dominant133 rld 2 recessive

Tomato 45 rld recessive for high shoot-formation capacity75 rc 2 dominant for regeneration capacity

Triticale 119

+ 2 for regenerationWheat 3

+ a few with major effect137

+ 2 (or few) dominant for green plant %12

+ 1 dominant for callus growth61

+ 232

+ at least 2 recessive

& Julien, 1992), cucumber (Nadolska & Malepszy,1989), maize (Bruneau, 1985 ; Hodges et al ., 1986 ;Rhodes et al ., 1986; Barloy et al ., 1989), orchard-grass (Gayin et al ., 1989), Solanum phureja (Cheng &Veilleux, 1991) and tomato (Koornneef et al ., 1987),dominant genes seem to control somatic tissue cultureability, or anther culture ability (Taylor & Veilleux,1992). Few reports suggest that complementary genesare necessary for the whole tissue culture process (Wanet al., 1988 ; Nadolska & Malepszy, 1989 ; Hernadez &Christie, 1990 ; Rakocszy-Trojanowska & Malepszy,1993; Yu & Pauls, 1993) .

The observation that embryogenesis and regen-eration abilities are controlled by a limited numberof genes is also confirmed by the fact that only areduced number of mutants are presently available inthe embryognic development of various species : car-rot, Arabidopsis thaliana and maize. `It seems reason-able to assume that the number of genes necessary forsuccessful plant development may be fairly limited'(Jurgens et al ., 1991) .

c) Gene location and determination

Addition, substitution and translocation wheat lines,and near isogenic lines (monosomic series, ditelo-somics, nullisomic-tetrasomics) were used to locatethe genes controlling high regeneration ability on thedifferent chromosomes (Table 2) . As early as 1975,experiments by Shimada & Makino established thatthere were significant differences in callus forma-tion from anther filaments between several ditelosomiclines (DT) and Chinese Spring (CS) . In addition, dif-ferences between CS and DT in in vitro somatic cal-lus growth were also described, suggesting that sever-al chromosome arms and hence genes were involved(Baroncelli et al., 1978) . Genes increasing microsporeembryo induction rate, regeneration ability and albinofrequency have been located on wheat chromosomes(Zhang & Li, 1984 ; Henry & De Buyser, 1985 ; Sza-kacs et al ., 1988 ; Agache et al ., 1989; De Buyser etal ., 1992b ; Gahemi & Sarrafi, 1994) . Mathias & Fukui(1986a), Galiba et al . (1986), Kaleikau et al . (1989a,1989b), De Buyser et al . (1992b) and Henry et al . (1993and in press), reported that particular chromosomesor chromosome arms controlled in vitro somatic andgametophytic embryogenesis and plant regeneration .IBS and 2DS wheat chromosome arms possess genesrequired to maintain embryogenic capacity over long-term cultures (Henry et al ., in press) . A review (Table2) suggests that competence for induction or mainte-

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nance of embryogenic callus and plant regenerationare both determined by a polygenic system involvinggenes with major and minor effects. As an example, allthe 4B chromosome substitutions into Chinese Springwheat cultures resulted in significant increases in shootregeneration (Mathias & Fukui, 1986a; Mathias et al .,1986b; Higgins & Mathias,1987 ; Mathias & Atkinson,1988 ; Mathias et al ., 1988) .

The next step was to map, on the chromosomes,the genes involved in tissue culture competence . Thiswas achieved by comparison of the recombinationbetween morphological, RFLP and RAPD markers andtissue culture ability. In such analysis, DNA probesco-segregating with genes conditioning tissue cultureability were identified . Recently RFLP probes linked togenes conferring high-androgenic capacity (Bentolilaet al ., 1992; Cowen et al ., 1992; Wan et al ., 1992), high-somatic embryogenesis ability (Armstrong et al ., 1992 ;Yu & Pauls, 1993), or shoot regeneration (Koornneef etal ., 1993; Komatsuda et al., 1993) have been identifiedin maize, tomato, alfalfa and barley.

The Table 2 results show that for wheat and maize,genes involved in anther culture ability are differentfrom genes involved in somatic tissue culture accord-ingly to the mendelian genetic studies detailed pre-viously (Table 1). The experimental results also con-firmed that gene location was different for the same dif-ferentiation process (i .e . regeneration ability) in somat-ic vs gametic tissue cultures, as well as for variousprocesses (embryo production and regeneration rate)for the same gametic tissue .

Surprisingly, the results shown in Table 2 are notconsistent with the conclusion of the mendelian geneticstudies which hypothetised that only few genes controlthe in vitro response . In fact, mendelian genetic studiesreveal only allelic differences between two genotypeswhich do not exclude that more genes are involvedwhen other genotypes are considered . Aneuploid anal-yses and RFLP studies also suggested the presence ofquantitative differences for tissue culture ability .

Research is currently directed towards isolation ofthe genes involved in tissue culture ability and regen-eration. The mapping of genes controlling particularsteps of the tissue culture process might allow thetransfer of them by sexual crosses to non respon-sive genotypes and characterize and identify thosegenes at the molecular level . The objective of severalresearch groups is to determine the underlying mecha-nisms responsible for tissue culture competence . Suchinvestigations (Reynolds & Kitto, 1992) will provideinsights both into mechanisms involved in embryo-

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Table 2 . Chromosomes and chromosome arms involved in tissue culture response of wheat, maize, tomatoand barley

genic gene regulation and into the molecular basis ofplant development.

Several laboratories have indicated that particulargenes are clearly involved in the somatic embryo-genesis process . Mathias & Fukui (1986a), Math-ias and Atkinson (1988) suggested that allelic vari-ation in wheat at the rht /gai gene (i .e . reducedheight/gibberellic acid insensitivity) might affect callusgrowth, somatic embryogenesis and plant regenerationperformances, via an effect on hormonal metabolism .More recently, Ben Amer at al . (1992b) indicated thatthe semi dwarfism gene rht8 and that the allele ppdl(day-length sensitive) had, respectively, a minor andmajor effect on wheat callus growth and regenerationability . Using rye, Linacero & Vazquez (1992) sug-gested that `the factor implicated in the development of

Ref # = literature reference number; EP = embryo production ; PR = plant regeneration ; AF = albinaformation ; CI = callus induction ; EM = embryogenesis. ( ) = chromosome or chromosome arm with minoreffect .

supernumerary embryos in vivo (polyembryonic seeds)also increased the in vitro somatic embryogenesis' . Inpotato, the correlation between the frequency of unre-duced pollen grains and anther culture capacity wasshown to be significantly negative (Meyer et al ., 1993),which suggests that genes reducing embryo production(or regeneration capacity) are linked to genes control-ling the first division restitution mechanism .

The influence of cytoplasmic genomes

The influence of cytoplasmic genomes cannot be stud-ied as easily as those of the nucleus . In the past, signif-icant differences for in vitro response were sometimesidentified in reciprocal crosses which do not necessar-

Species Ref # Anther culture Somatic culture

EP PR AF CI EM PR

Wheat 56, 96, 97 IRS3, 35, 2085, 36, 583 1D,5BL 5B 5B132 1 B, 1 D, 2A, 3A et 4D (2D,

5B, 7A 3B, 3D, 6B)149 2A et 2D (2B,

4A,5A,5B)127 4BL77 IRS46 7B,7D, ID43 IBS, 2BS, 6BL65,66 2AL, 2BS,

88,612BL, 2DL4BL

91,9213 2DL 2DL59 1AL, 3AL, 3BL, 3DL,

Maize 14 3,9(1BL, 3DS)

29 3L, 9, (1, 10)4 9L

9L143 2L, 8L 1, 2, 3 et 6

TomatoBarley

7674

3 'SR'2 'Shdl'

ily indicate the presence of cytoplasmic gene effects,since maternal effects may be involved . Thus it is nec-essary to discriminate between the two phenomena :- maternal effect : the maternal parent can influencethe phenotype of the progeny, regardless of its geno-type. This does not involve the cytoplasmic DNA ofthe progeny.- maternal (cytoplasmic to be more accurate) inher-itance . In this case, only the maternal chloroplas-tic and mitochondrial DNAs are transmitted to theprogeny and are susceptible to modify its phenotype .Most species do not transmit functional cytoplasmicorganelles through the pollen . In this case, reciprocalF, crosses should be complemented by backcross orF2 segregations and molecular analysis to demonstratecytoplasmic inheritance .

Our results with F, wheat hybrids indicated thatfew significant reciprocal effects for embryo produc-tion and regeneration ability can be revealed for bothanther (Henry & De Buyser, 1985, 1990) and somatic(De Buyser et al ., 1992a) tissue cultures . The absenceof reciprocal differences was also shown with otherspecies both in anther culture experiments (Bullok etal., 1982; Dunwell et al ., 1987; Quimio & Zapata,1990; Larsen et al ., 1991 ; Zhou & Konzak, 1992) or insomatic tissue culture experiments (Frankenberger etal ., 1981) . In some cases, differences between recip-rocal crosses were most likely to be due to samplingerror, small numbers of regenerants, or heterozygos-ity of the mother plants (Raquin, 1982), rather thangenetic effects . In wheat, the lack of significant differ-ences between reciprocal crosses is not really surpris-ing considering that cytoplasms of most varieties arenot different for mtDNA and cpDNA . So far, Triticumaestivum cytoplasm was even considered as `unique' .Most wheat varieties should be therefore consideredas euplasmic, i .e. different nuclear DNA contents withthe same cytoplasm. The differences observed in recip-rocal crosses involving wheat cultivars with similar Taestivum cytoplasm suggests therefore the influence ofa maternal effect. The presence of reciprocal differ-ences (Nesticky et al., 1983; Beckert & Qing, 1984 ;Powell, 1988 ; Ekiz & Konzak, 1991c) among crossesis generally not sufficient to conclude that cytoplasmicgenetic factors were involved . Cytoplasms have also tobe differentiated using a molecular approach .

A few wheat varieties possess cytoplasms otherthan T aestivum, for example T ventricosum, T tim-opheevi . Multiple alloplasmic lines with the `same'nuclear genome in several alien cytoplasms have beendeveloped by recurrent back-crosses .

5 1

- As early as 1973, using anther culture of cultivarChamplein with T timopheevi cytoplasm, Picard & DeBuyser (1973) reported successful haploid plant regen-eration. Later T timopheevi cytoplasm was reported toincrease wheat and triticale embryo production (Picardet al., 1977 ; Charmet & Bernard, 1984; Becraft & Tay-lor, 1989, 1992) during anther culture . T kotschyi andT juvenale cytoplasms also increased embryo produc-tion (Sagi & Barnabas, 1989) . On the contrary, T spel-toides cytoplasm reduced embryo production (Becraft& Taylor, 1989). Triticale lines having T timopheevicytoplasm have a higher rate of differentiation com-pared to those possessing the T aestivum cytoplasm(Charmet & Bernard, 1984) . Triticale genotypes hav-ing rye cytoplasm possessed also higher embryo pro-duction (Gordey & Sorokin, 1991) . Using reciprocalcrosses between wheat and alloplasmic lines, cyto-plasm and cytoplasm x nuclear genetic control ofanther culture competence were demonstrated (Becraft& Taylor, 1989; Ekiz & Konzak, 1991b) . Depend-ing upon the cultivar nuclear genome used (CS, Butte,Selkirk, Penjamo 62, Siete Cerros 66) the cytoplas-mic effects were sometimes observed to be different.T speltoides and T ventricosum cytoplasms alwaysreduced embryo frequency . The nuclear genome ofthe cultivar Selkirk (T aestivum) reduced embryo fre-quency with any cytoplasm (Ekiz & Konzak, 1991 a) .On the contrary, cytoplasms from T peregrinum, Tlongissimum and T kotschyi have variable influenceson embryo frequency . T kotschyi, T ventricosum, Tmacha and T cylindricum cytoplasms also affectedplant regeneration (Sagi & Barnabas, 1989 ; Ekiz &Konzak, 1991a) .- Haploid wheat embryo production through cross-es with Hordeum bulbosum is increased by T ovatumcytoplasm (Ozdemir et al ., 1990) .- Effects of alien cytoplasms have also been observedduring somatic wheat tissue culture, influencing spe-cially callus induction (Orlov & Pavilova, 1989 ; Math-ias & Fukui, 1986a), callus growth (Kinoshita & Mika-mi, 1984) and somatic embryogenesis ability (Mathiaset al ., 1986b; Felsenburg et al ., 1987). For example,T timopheevi cytoplasm improved plant regenerationfrom scutellar calli (Felsenburg et al ., 1987) and thecytoplasm of T ovatum decreased leaf primordia for-mation and affected the response of callus cells to 2,4-D(Mathias & Fukui, 1986a) . On the contrary, Mathiaset al . (1986b) stated that none of the cytoplasms testedmodify plant regeneration when associated with the CSnucleus .- Significant differences between cytoplasms were also

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described in Brassica carinata for shoot morphogen-esis (Narasimhulu et al ., 1989), in maize for callusgrowth (Nesticky et al., 1983) and somatic embryoge-nesis (Tomes, 1985 ; Willman et al., 1989), in barley foranther culture responses (Powell, 1988), in potato forandrogenetic competence (Singsit & Veilleux, 1989)and in Medicago species for somatic embryogenesis(Walton & Brown, 1988) and regeneration (Wan et al .,1988) .

The presence of maternal effects and cytoplasmicinheritance during interspecific crosses (Worland et al .,1988) converge with observations that particular mito-chondria) genome organizations are correlated with thedifferent regeneration ability of somatic tissue cultivat-ed in vitro (Rode et al ., 1988) .

Genetic information in the nucleus regulates theexpression of the mitochondrial genome as well asthe expression of nuclear genes encoding mitochon-dria) proteins . For example, the homoeologous group1 chromosomes of Triticum sp. carry `species cyto-plasm specific' nuclear genes that alter developmentand reproductive traits in alloplasmic lines (Maan &Endo, 1991) . It is therefore possible that regenerationfrom tissue culture is at least partly controlled by spe-cific nuclear-cytoplasmic interactions .

Relevance to plant breeding

The choice of parental strains for a breeding programshould be realized on agronomic criteria rather thancompatibility with a particular tissue culture technique(Henry & De Buyser, 1990). The genetic variationobserved in tissue culture ability raises therefore, sev-eral questions concerning the involvement of suchtraits in plant breeding . First of all, it seems neces-sary to separate genes involved in short-term (haploidyand somatic embryogenesis) and long-term (somaticembryogenesis, cell and protoplasts techniques) tissueculture abilities, particulary when monocotyledonousspecies are considered .

Short-term tissue culture. For most species, it has beenpossible to observe large differences between geno-types for short-term tissue culture ability and thosedifferences are mainly quantitative . For anther culture,less than 5% of the breeders' wheat stocks that we test-ed failed to produce green plants (De Buyser & Henry,1979; Henry & De Buyser, 1990) . In fact, this tech-nique is effective for most wheat genotypes, but often

with laborious efforts . However, only a limited num-ber of genotypes has high responsiveness for in vitroculture .

The evidence available so far, leads to the conclu-sion that short term in vitro culture competences areheritable traits . Thus, it should be relatively easy if fewgenes are involved to incorporate regeneration capacityinto agronomically valuable genotypes . When a largerange of genotypes has been screened for in vitro tissueculturability, it is easy either to cross the most efficientones when the tissue culture character is additive (Hal-berg et al ., 1990) or to produce efficient x non efficienthybrids when it is dominant. Such experiments havebeen performed for barley anther culture using the Igricultivar, for Dactylis glomerata somatic embryogen-esis (Gavin et al., 1989), for alfalfa (Reisch & Bing-ham, 1980) and tomato regeneration (Koornneef et al .,1987). As early as 1981, Frankenberger et al . dial-lel experiments suggested that `crosses between highshoot-forming parents would result in progeny withhigh capacities for shoot formation' . In 1984, Beckert& Qing indicated `that it should be possible to select foraptitude to in vitro culture' . Becraft & Taylor (1992)indicated that a high heritability makes wheat andro-genesis an easy trait to manipulate. The intermating ofselected tissue culture-derived genotypes is an effec-tive means to increase in vitro responsiveness (Petolinoet al ., 1988 ; Barloy et al ., 1989) . We also know fromexperimental evidence that a high level of tissue cultureresponse could be obtained by crossing complemen-tary genotypes (Henry & De Buyser, 1985) . Attemptsto improve tissue culture response through mendeliangenetics have been successful in producing recombi-nant lines in which efficient genes were accumulated(Bingham et al ., 1975 ; Wenzel & Uhrig, 1981 ; Beck-ert & Qing, 1984 ; Henry & De Buyser, 1985 ; Agacheet al ., 1988 ; Petrolino et al ., 1988; Ray & Bingham,1989; Halberg et al ., 1990; Henry & De Buyser, 1990) .Nevertheless, improvement of tissue culture responsethrough breeding remains a difficult task due to multi-ple cycles of tissue culture and plant crosses requiredfor this process .

In several wheat anther culture experiments, wehave observed higher frequency of green microspore-derived plant production in hybrids than in the bestparents (Henry & De Buyser, 1985) revealing highpositive heterosis . Ouyang et al . (1983) obtained com-parable results and suggested that the use of F1 hybridsas anther-donors is an effective means of increasinggreen microspore-derived plant production . Unfortu-nately, for a lot of species, tissue culture traits are con-

trolled by recessive alleles. In those cases F2 (or BC1)plants need to be used. When more than two genes areinvolved for the control of tissue culture response, thetrait can be transferred through one or a few cycle ofselfing or backcrossing, like it was performed for wheatsomatic embryogenesis (De Buyser et al ., 1992a) . Thedevelopment of germplasm with enhanced levels of invitro culturability allowed significant progress in plantbiotechnologies and plant breeding .

Long-term tissue culture. Differences between geno-types for long-term tissue culture competence aremainly qualitative. For most of the major crops, thegenes controlling this competence are often present ina limited number of genotypes . For example, maizeembryogenic type II callus, long-term wheat somaticembryogenesis and `cell suspension' culture can onlybe obtained from few genetic combinations . The trans-fer of such abilities to a wide range of agronomicallyinteresting genotypes would require extensive work,time and money . This represents a strong limitation forimprovement of long-term tissue cultures, to a pointthat it marginalized protoplast fusion techniques anddelayed for years the development of genetic trans-formation techniques beneficial for plant breeding ofmonocots .

This limitation is more difficult to overcome thanpreviously reported for short-term tissue culture com-petence. However, using sexual crosses during longterm breeding programs, the `tissue culture controllinggenes' can be introduced in other germplasms withseveral flanking loci. The use of a recurrent breed-ing scheme will be particularly interesting with suchcharacters which present low selection efficiency (highenvironmental effects) or limited genetic gain . It is alsonecessary to evaluate carefully such breeding programswhen the tissue culturability is the result of specificcombining ability, considering the numerous test cross-es involved in such a process . In addition to the trans-fer of the `tissue culturability genes' by sexual crosses,genotype conversion to long-term tissue culture com-petence could be performed by genetic transformation .In the near future, genes controlling plant tissue cul-ture response will be identified and isolated . Thosegenes represent an opportunity to involve recalcitrantgenotypes for tissue culture through genetic transfor-mation . In a first step, transfer and expression of suchgenes should increase tissue culture capabilities . Thisshould be of particular importance considering thatmost genotypes in important crop species including

53

monocots (rice, wheat, maize, barley) are recalcitrantfor tissue culture. In a second step it will be possible tointroduce genes of agronomic interest into genotypesmodified for tissue culture competence .

Conclusion

Plant regeneration through in vitro tissue cultureremains necessary for most genetic manipulation ofcrop plants and subsequent benefits for plant breed-ing. Haploidisation, somatic embryogenesis, cell sus-pension culture and protoplast techniques remains theprincipal technologies requiring plant regeneration forfuture involvement in plant breeding . Genetic progressis expected from those technologies both by savingtime and increasing genetic variation .

The traits for somatic embryogenesis and antherculturability are sexually heritable. Numerous evi-dence demonstrates that genetic factors, mostly nucleargenes, condition in vitro tissue culture response . Genet-ical differences are potentially valuable for analyz-ing physiological and biochemical differences amonggenotypes for tissue culture capacity . Despite all theefforts attempted in this domain, detailed knowledgeconcerning the genetic control of plant regenerationremained limited . The most detailed studies on thegenetic control of pollen embryogenesis were per-formed on maize and to a lesser extent on wheat dueto its allohexaploid genetic status .

Very little is known about the molecular eventsthat trigger tissue culture capacity (Lindsey & Top-ping, 1993). A common misunderstanding would be toattribute tissue culturability to `tissue culture genes' .It is obvious that genes involved in the tissue cul-ture response were not primarily present in the plantgenome for this purpose . Several non genetic fac-tors such as environmental and tissue culture condi-tions also clearly influence the in vitro culture process .The predominant role of hormones in plant growthand development suggest that hormone involvementin tissue culture competence is of major importance(Close & Gallager, 1989 ; Arnison et al ., 1990 ; Izhar& Power, 1977) . The genetic basis of tissue cultura-bility could therefore rely on genes involved in planthormone metabolism . Nevertheless, the role of hor-mones in somatic or haploid embryogenesis remainsnot fully understood . Auxins are the most impor-tant factor for embryogenesis induction and develop-ment. Mathias & Fukui (1986a) suggested that the 4Bwheat chromosome effect on somatic embryogenesis

54

might correspond to a modification of the cellular hor-monal metabolism which alters the cell sensitivity toexogenous hormone. It can be hypothesized that genesinvolved in the phytohormone signals are involved inplant regeneration . Several protein kinases were report-ed as signalling molecules for growth factors and hor-mones. Other types of molecules are likely to be impor-tant signals : for animal cells the distribution of intra-cellular calcium (second messager) is involved in asignal transduction chain, a cascade, which activatesprotein kinases controlling genes acting on differenti-ation. Moreover, calcium is associated in Fucus withthe maintenance of polarized growth of the embryo .It is a reasonable working hypothesis that comparabletransduction chains may be operative in plant embryo-genesis in vitro as well as in situ . The genes involvedin such processes are largely unknown, but would haveto be active very early in the development .

The development of a whole plant from a single cell(egg, somatic, microspore) `requires both the deter-mination of many cell types and the organization ofthese cells into an elaborate pattern' (St Johnston &Nusslein-Volhard, 1992). Komamine et al . (1992) andReinbothe et al . (1992) suggested that `only a few pro-teins play important roles during embryogenesis' . Itwas estimated that a relatively small number of genesmay be sufficient to control pattern formation in theArabidopsis thaliana embryo . This number convergeswith the estimations of the control of patterning in theDrosophila melogaster embryo (Lindsey & Topping,1993 ; St Johnston & Nusslein-Volhard, 1992; Mayeret al ., 1991 ; Jurgens et al ., 1991) . This is also consis-tent with the limited number of genes shown by geneticstudies, to be involved in in vitro culturability (Table2) .

All genotypes possess necessary genetic informa-tion for in vivo zygotic embryogenesis . The variationfor in vitro culture responses depends therefore, eitheron the regulation of genetic information, or on theability of plant cells to enter, at different developmen-tal steps, a new developmental program . Consider-able progress is expected from identifying the so-called`regeneration genes', or their protein products (Pechanet al ., 1991 ; Vergne et al ., 1993) .

The study of plant regeneration should be focusedon early molecular signals in systems available forgenetic and biochemical analyses (somatic, microsporeand zygotic embryogenesis) . Molecular approaches(Reynolds & Kitto, 1992) of early events triggeringmorphogenesis and differentiation of plants need to beinvestigated . It seems also that the understanding of

tissue culture ability will require consideration of bothcytoplasmic and nuclear genomes .

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