A procedure for achieving plant regeneration from inflorescence-derived callus cultures of hexaploid triticale was developed. Genotypic differences.
Plant Science Letters, 24 (1982) 275--280 Elmvier/North-Holland Scientifm Publishers Ltd.
275
PLANT REGENERATION FROM INFLORESCENCE CULTURES OF HEXAPLOID TRITICALE*
C. NAKAMURA** and W.A. K E L L E R t Ottawa Research Station, Research Branch, Agriculture Canada, Ottawa, Ontario K I A
OC6 (Canada) (Received April 28th, 1981) (Revision received August 18th, 1981) (Accepted September 10th, 1981) SUMMARY
A procedure for achieving plant regeneration from inflorescence-derived callus cultures of hexaploid triticale was developed. Genotypic differences in the frequency of totipotent callus formation were evident. Calli initiated on agar had a higher morphogenic capacity than those initiated on liquid medium. No growth regulator re~mes enhanced the morphogenic response, except for root induction which was stimulated by gibberellic acid (GA3) or triiodobenzoic acid (TIBA). Although the frequency of totipotent callus induction was low, large numbers of plants could be ~btained from individual calli. The regenerated plants were generally shot ~er and less vigorous than the original donor material, but the majority of those examined had the expected euploid chromosome number indicating that their altered morphology had a physiological rather than a genetic basis. INTRODUCTION
Plant regeneration has been induced in inflorescence or rachis explant cultures of a number of graminaceous species including Sorghastrum nutans [1], Andropogon gerardii [2], Saccharum spp. [3--5], Triticum spp. [6--8] Zea mays [9,10], Sorghum bicolor [11] and Triticum crassum X Hordeum vulgate hybrids [8]. We selected hexaploid triticale as a model cereal for studies on morphogenesis in vitro and have obtained high frequencies of plant regeneration from immature embryo-derived callus cultures of this *Contribution No. 648 Ottawa Research Station. **Present address: Laboratory of Genetics, Faculty of Agriculture, Kobe University, Kobe, Nadaku, Rokkodai 1, Japan 657. t T o whom reprints request should be sent. Abbreviations: BAP, 6-benzylamlno purine; GA~, gibberelllc acid; 2,4,5-T, 2,4,5trichlorophenoxyacetic acid; TIBA, 2,3,6-triiodobenzoic acid. 0304--4211/82/0000--0000/$02.75 © Elsevier/North-Holland Scientific Publishers Ltd.
276 species [12]. To extend the availability of suitable explant sources, we initiated a study on morphogenesis in callus derived from immature inflorescences of triticale. In this communication we report on the significance of cultivar type, physical composition of the medium and growth regulator t y p e and concentration on morphogenesis in such cultures. MATERIALS AND METHODS Plants of three hexaploid triticale (× Triticosecale Wittmack) cultivars, Beagle, Rosner and Welsh, were grown in chambers at a day/night temperature of 20°/15°C under a 16-h photoperiod. Light was supplied by a combination of fluorescent and incandescent source at an intensity of 32 000 lux. Explants for callus induction were obtained from 1--3-cm immature inflorescences within the flag leaf sheaths. After surface sterilization according to methods described previously [ 1 2 ] , the inflorescences were transversely sectioned and plated on agar (0.8% Difco Bacto) or floated on liquid medium in 60 × 15 mm plastic petri dishes. The basal medium used was Kao's medium [13] modified as described previously [ 12]. F o r callus induction the basal liquid medium was supplemerited with 1--10 mg/1 of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T); explants were incubated for 4 weeks at 24°C in a 10-h photoperiod under fluorescent light (500 lux). An auxin concentration of 5 mg/1 was used for callus induction on solid medium. Calii induced on liquid were subcultured once on liquid medium containing 2 mg/1 of 2,4,5-T under the same conditions initially employed for callus induction. Calli induced on agar were subcultured in the same manner b u t were consistently maintained on solid medium. Attempts to induce morphogenesis involved the transfer of subcultured calli to liquid or solid media (60 × 20 mm petri dishes) which were either free of, or supplemented with growth regulators. Types and concentrations of growth regulators tested in shoot induction included the auxins 2,4,5-T, 2,4-dichlorophenoxyacetic acid and a-naphthaleneacetic acid all at 0.01, 0.1, 1 and 2 mg/l, the cytokinins kinetin, zeatin, 6-benzylamino purine (BAP) and 6-7-7-dimethylallylamino purine all at 1 and 5 mg/1, GA3 at 1, 5 and 10 mg/1 and TIBA at 1, 5 and 10 mg/1. Auxins were added to the medium alone or in combination with a cytokinin or GA3. The antiauxin TIBA was also used alone or in combination with a cytokinin. Calli were incubated under continuous fluorescent light (1500 lux) at 25°C for 4-week periods. The precedures employed for plant regeneration and cytological characterization have been previously described [ 12]. RESULTS Callus induction from inflorescence explants occurred within several days on both liquid and solid medium. Calli induced on solid medium w e r e
277 yellowish and c o m p a c t while those on liquid medium were whitish and fragile. R o o t initiation occurred frequently during the callus induction period. The frequency of callus induction depended on auxin concentration and physical composition of the medium. An optimal 2,4,5-T concentration for callus induction in liquid was 2--5 rag/l; liquid was superior to agar for callus induction at 5 rag/1 2,4,5-T (Table I). Calli induced on liquid usually ceased to grow and often became necrotic upon transfer to solid medium whereas those induced and maintained on solid medium continued to grow. Induction of morphogenesis was achieved by transferring subcultured calli to regeneration media. Calli induced on liquid medium did not undergo morphogenesis, except for root formation, either on liquid or solid regeneration medium during the first 4-week induction period. Shoot differentiation was achieved after at least three successive subcultures on solid regeneration medium free of, or supplemented with various concentrations and combinations of growth regulators. Shoot differentiation occurred in 4.7% and 1.6% of the inflorescence explants of Welsh and Rosner, respectively, b u t was n o t achieved in Beagle (Table II). S h o o t regeneration capacity in calli induced on liquid medium was neither enhanced nor depressed by any growth regulator treatments employed. Morphogenic cultures when obtained, however, regenerated many plantlets during successive culture periods. Calli induced on solid medium responded to the morphogenic induction at a much higher frequency. After two successive cultures on solid regeneration medium either free of, or supplemented with growth regulators, shoot regeneration was achieved from 19.0%, 6.1% and 5.4% of the inflorescence explants in Welsh, Rosner and Beagle, respectively (Table III). Growth regulator regimes which enhanced shoot differentiation over that obtained with hormone-free medium were not identified. The addition of BAP or kinetin to the basal medium induced necrosis in most calli during the early subcultures. The
TABLE I EFFECT OF 2,4,5-T CONCENTRATION ON CALLUS INDUCTION FROM IMMATURE INFLORESCENCES OF HEXAPLOID TRITICALE CULTURED ON LIQUID OR AGAR-SOLIDIFIED KAO MEDIUM 2,4,5-T
Physical
conc.
compo~tion
(rag/l)
% induction
No. of inflorescences
Beagle
Rosner
Welsh
Average
1 2 5
Liquid Liquid Liquid
33 91 70
53 63 75
57 74 73
48 76 72
54 217 300
5 10
Solid Liquid
68 58
52 36
53 50
58 45
315 49
Average
Liquid
72
66
70
278 TABLE H MORPHOGENIC RESPONSE IN LIQUID MEDIUM-INDUCED INFLORESCENCE CALLI OF HEXAPLOID TRITICALEa Cultivar
No. of inflorescences
No. of morphogenie inflorescencesb
No. of plants regenerated c
Beagle Rosner Welsh
179 190 148
0 3 (1.6%) 7 (4.7%)
0 7 90
aInduetion of morphogenesis was carried out on egar-solidified Kao medium free of growth regulators, or supplemented with various concentrations and combinations of growth regulators (see Materials and Methods). bInflorescences from which morphogenic callus cultures were induced. cSeven plants were regenerated from one Rosner inflorescence. Sixty-seven regenerates were obtained from one Welsh inflorescence and 23 green plants as well as many albinos and chimeras were obtained from a second Welsh inflorescence. Many shoots differentiated from the 7 remaining inflorescence cultures but attempts were not made to regenerate plants.
differentiation of roots on the callus surface was enhanced by the addition o f 1--10 mg/l GA3 or TIBA. The majority of regenerates were green but some albinos and chimeric plantlets were obtained from liquid-induced calli during later regeneration periods. The regenerates were shorter, underwent less tillering, and set fewer seeds than the initial d o n o r material. R o o t tip chromosome counts of 10 r a n d o m l y selected Welsh regenerates revealed 9 plants with the euploid chromosome number of 42 and one with 41 chromosomes.
TABLE HI MORPHOGENIC RESPONSE IN SOLID MEDIUM-INDUCED INFLORESCENCE CALLI OF HEXAFLOID T R 1 T I C ~ a Cultivar
No. of inflorescenees
No. of morphogenie inflorescencesa
Beagle
111
6 (5.4%)
Rosner Welsh
99 105
6 (6.1%) 20 (19.0%)
No. of plantlets regenerated b
-17 174
aSee footnote for Table H. bSeventeen plantlets were regenerated from 3 Roener inflorescences and 174 plantlets were regenerated from 10 Welsh inflorescenees. The remaining morphogenic cultures derived from 19 inflor~eenees regenerated many shoots which were neither counted nor used for plantlet regeneration.
279 DISCUSSION
In this study we have demonstrated the totipotency of inflorescencederived triticale callus cultures (Tables II and III). Although the frequency of totipotent callus induction was low, many plants could be regenerated from individual calli. However, the regenerates were generally shorter with fewer tillers and lower fertility than the original cultivars. Chromosome counts of 10 randomly selected Welsh regenerates revealed that the aneuploid frequency was in agreement with that observed in the original cultivar [ 12,14]. It is therefore probable that the morphological abnormalities we observed were mainly physiological and that, in triticale, inflorescencederived regenerates are more stable in their chromosome constitution than the immature embryo-derived regenerates initially studied [12]. Somatic chromosome stability was also observed in inflorescence-derived regenerates of a T. crassurn X H. vulgare hybrid, although T. crassum regenerates exhibited a consistent loss of chromosomes [8]. Inflorescence culture techniques should have potential in the clonal propagation of cereal species, particularly of sterile interspecific hybrids and they may also be useful in the development of morphogenic haploid tissue culture systems for use in mutant selection. The cultivar Welsh underwent higher frequencies of shoot induction than the other cultivars, irrespective of medium composition (Tables II and HI) and growth regulators employed. The superiority of this cultivar in morphogenic competence was also demonstrated in embryo-derived callus cultures and may be related to its chromosomal substitutions [12]. Welsh and possibly other substitutional triticales in which rye chromosomes are replaced by corresponding D genome chromosomes of common wheat should be of value for further tissue culture studies in this synthetic allopolyploid crop species. Kao's medium [13] was found to be suitable for the clonal propagation of hexaploid triticale through immature inflorescence culture. The superiority of this medium over Bs [15] and MS [16] media was shown in a previous study of immature embryo-derived callus cultures [12]. We have recently observed that a modified Kao medium free of casamino acids and coconut milk was also capable of sustaining growth and competence for plant regeneration in immature embryo-derived suspension cultures and in seedling epicotyl-derived callus cultures of the cultivar Welsh (unpublished). Although in the present study no particular growth regulator regimes were found to be superior for shoot regeneration, efforts will be continued to develop a more effective medium for cereal tissue culture studies. ACKNOWLEDGEMENTS C. Nakamura held a Visiting Fellowship from the Natural Sciences and Engineering Research Council of Canada and the work was carried out with the support of Agriculture Canada, to both of which he is indebted.
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