Micropropagation of rhubarb with special reference to weaning stage and subsequent growth Carola Lassus and Irma Voipio Lassus, C. & Voipio, I. 1994. Micropropagation of rhubarb with special refer- ence to weaning stage and subsequent growth. Agricultural Science in Finland 3: 189-194. (Department of Plant Production, P.0.8. 27, FIN-00014 University of Helsinki, Finland.) Micropropagation of rhubarb (Rheum rhabarbarum L.) on MS medium with su- crose 20 g/l and agar 8 g/l, supplemented with benzylaminopurine (BAP) 1 mg/1 and indolebutyric acid (IBA) 1 mg/1 for initiation and multiplication, was studied with cv. Victoria and clone AF. With clone AF, the effects ofrooting method (direct rooting, direct rooting with Floramon A or in vitro rooting) and propagule size (height 1.5-2.0, 2.1-5.0 or 5.1-10.0 cm) on weaning survival and plant size were examined. Further growth was recorded over a 3-year period in field studies. The medium gave a multiplication rate of 3.2/4 weeks for clone AF. For cv. Victoria, the medium did not seem suitable because of high occurrence of callus and vitrification. The multiplication rate of cv. Victoria was, however, increased from 2.8 to 5.4/4 weeks by using propagules from non-sprouting instead of sprout- ing buds. The weaning survival of clone AF averaged 86%. Rooting method did not affect either weaning survival or plant size. Propagule size affected plant size, but not weaning survival or further growth in the field. Key words: direct rooting, in vitro rooting, growth in field studies, propagule size Introduction Rhubarb is usually propagated vegetatively since seed propagation results in undesired variation among progeny. Conventional propagation by crown division gives, at best, a 4- to 6-fold mul- tiplication rate every 2 years. By using single- bud division, ca. 240 plants/mother plant can be produced during a 6-month season (Case 1970, Norman 1978). Viruses are, however, transmit- ted through these propagation methods. Walkey Abbreviations: MS = Murashige and Skoog (1962), BAP = benzylaminopurine, IBA = indolebutyric acid, NAA = naphthaleneacetic acid (1968) succeeded in eliminating viruses from rhu- barb through in vitro culture of meristem tips. The method has been further developed for rapid multiplication (Roggemans and Claes 1979, Walkey and Matthews 1979, Pierik et al. 1989, Camara Machado et al. 1990,Rumpunen 1990). In theory, based on the average multiplication rate of 2.8/2 weeks obtained by Walkey and Matthews (1979), it is possible to produce over a million rhubarb plants within 7 months from a single meristem tip initiated in vitro. In micropropagation, direct rooting is recom- mended whenever possible because of considera- bly lower costs compared with in vitro rooting. Furthermore, roots formed in vitro are easily dam- 189 Agricultural Science in Finland 3 (1994)Research Note https://www.c-info.fi/en/info/?token=YHfOugSAxFhbNaNf.4Pks1rboRhk4raCc8BkdHQ.m1AiVqetZAQbWjnWlow2IWmYvd2YqH0kN0y36zW8sY7bQV9Mx7rJmtgnQzEn4BTYA2EJ8qQ-ktD1Rh0jFTRKzyNf7FdVhnIAvcnwvjQyg0H70efaeechC-7A78cxPaLd-H-CJGongq379Za8afhnEEwpfj29yjmXtI4Z_lDKUDC8hwrrFiOAZg9NQVZj5K5JFdxvLjThYPXsqcKCIPsiMto1jriXgB1lvbfUQi4HLWejKCqUIWryQB-jcw2NfSBtCSJu9VqcCNM7dBfWjGByT4SkloY aged during planting (Conner and Thomas 1981, Debergh and Maene 1981). To maximize wean- ing survival, it appears that the propagule must exceed a certain minimum size (Conner and Tho- mas 1981). The leaves formed in vitro are found to act mainly as storage organs (Wardle et al. 1983) and, after transferring to soil, enough nu- trient reserves should be present to supply the plant’s requirements until total autotrophy is reached (Capellades et al. 1990). The aim of this study was to investigate mi- cropropagation of rhubarb, especially the effects of direct rooting and propagule size on weaning survival and ex vitro growth. To observe the after effects, further growth was examined in a field trial over 3 years. Material and methods Clone AF was used as plant material in Experi- ments 1 and 4, and cv. Victoria in Experiments 2 and 3. In Experiment 1, the shoot multiplication rate was calculated from 139 propagules and in Experiment 2 from 70 propagules in the third subculture. In Experiment 1, the buds available were mostly non-sprouting and in Experiment 2 mostly sprouting. In Experiment 3, the multipli- cation rates of propagules from sprouting and non- sprouting buds (13 and 24 propagules, respec- tively) were compared in the first subculture. In Experiment 4, where the shoots of clone AF from Experiment 1 were used, the effects of root- ing method (in vitro rooting, direct rooting and direct rooting with Floramon A treatment (0.1 % NAA)) and propagule size (small, medium and large) on weaning survival and plant size ex vitro were studied. The propagule size categories, meas- ured as shoot height on the basis of the longest petiole, were 1.5-2.0cm (small), 2.1-5.0 cm (me- dium) and 5.1-10.0 cm (large). In Experiment 4, the factor combinations were arranged in tripli- cate (with 10 plants per replicate) according to a randomized block design. Experiments 1 and 2 began with in vitro initia- tion in May and Experiment 3 in October 1989. The excised buds from parental crowns were washed in tap water, disinfected in 3.5% sodium hypochlorite for 10 min and rinsed 3 times in sterilized, deionized water. The excised shoot tips were 1-2 mm in diameter. The basal medium consisted of MS medium with sucrose 20 g/1 and Bacto agar (Difco) 8 g/1, with pH adjusted to 5.5. For initiation and multiplication, the media were supplemented with BAP (Sigma) 1 mg/1 and IBA (Merck) 1 mg/1. No growth regulator was added to the rooting medium. All media were autoclaved for 15 min at 121°C. The explants were initially grown in culture tubes containing 10 ml medium and, after 4-6 weeks, were transferred to 100 ml Erlenmeyer flasks containing 50 ml medium. The multiplica- tion stage was started after an initiation period of 8 weeks (Experiments 1 and 2) or 6 weeks (Ex- periment 3). The temperature in the growth cham- ber was 22-27°C, except during the multiplica- tion stage in Experiments 1 and 2, when it occa- sionally rose to 31°C. Daylength was 16 h and irradiance ca. 6 W nr2 (PAR) (lamp type ‘Kirkas de Luxe’, Airam). In Experiment 4, the shoots for in vitro rooting were kept for one week on the rooting medium. At transplanting, 32% of the shoots had 1-10 visible roots 0.1-5.0 cm in length. The shoots for direct rooting were harvested directly from the multiplication medium, and half of the shoots were treated with Floramon A. For transplanting, all shoots were transferred to a glasshouse and placed in a plastic tent which was shaded during sunny days. The growth substrate was a 2:1 mixture of fertilized peat (Vapo B2) and vermiculite (grain size 2-3 mm). The mean air temperature in the glasshouse was 22°C. Natural light conditions pre- vailed at the start of the growing period (Septem- ber), and after 5 weeks supplementary light was provided for 16 h/day (lamp type HPI-T, Philips). After 18 days, the relative humidity was reduced from 100% to ca. 70% over a period of one week by gradually opening the tent. At the same time, the irrigation water was supplemented with grad- ually increasing amounts (0.05-0.2%) of fertiliz- er. During the first 3 weeks, the fertilizer was ‘4- Superex’ (17N-4P-25K, Kekkilä), and thereafter ‘5-Superex’ (11N-4P-25K, Kekkilä). 190 Research NoteAgricultural Science in Finland 3 (1994) AgriculturalScience in Finland 3 (1994) Plants rooted in vitro and those rooted directly without Floramon A (all size categories: 2 x 3 x 8 = 48 plants) were potted and overwintered in a glasshouse until May 1990 when they were plant- ed randomly in an outdoor nursery at a density of 30 cm x 30 cm. On 29 May 1991, the plants were transplanted in a field at a density of 100 cm x 125 cm. Conventional growing techniques were adopted. The first crop was harvested in 1992. The field experiment was arranged in quadrupli- cate (with two plants per replicate) according to a randomized block design. In Experiments 1 to 3, the number of shoots/ propagule was counted after 4 weeks. Callus oc- currence was recorded in Experiments 1 and 2, and vitrification in Experiments 1 to 3. The mul- tiplication data from Experiment 3 were analyzed by Mann-Whitney’s U-test. In Experiment 4, the proportion of surviving plantlets, the length of the longest petiole, the number of leaves, and fresh and dry weights of the foliage were record- ed. In the field trial, the number of leaves, and the length and the weight of the longest petioles at harvest were recorded. A logistic regression model and analysis of variance were applied to the survival results. The other data were analyzed using analysis of variance and the S-N-K test for mean separation. Results In Experiment 1 with clone AF, the multiplica- tion rate was 3.2/4 weeks, and in Experiment 2 with cv. Victoria 2.1/4 weeks. During initiation, callus occurrence was 30% for clone AF and 80% for cv. Victoria, and during the multiplication stage 78 and 100%, respectively. No vitrification was found in clone AF, but in cv. Victoria, 15% of the shoot clusters were vitrified. In Experiment 3 with cv. Victoria, the multiplication rate was higher (p<0.05) for propagules from non-sprouting buds (5.4/4 weeks, variation interval [1,17]) than from sprouting buds (2.8/4 weeks, [l,B]). In both treat- ments, 35% of the shoot clusters were vitrified. In Experiment 4, no significant differences in weaning survival or plant size caused by differ- ent rooting methods were found (Tables 1 and 2). Propagule size did not affect weaning surviv- al significantly, but plant size was affected (Table 1). After a 7-week growing period, plants from large propagules had a dry and fresh weight 4.5-fold higher than those from small propagules (Table 2). Except for one of the in vitro rooted plant of the category “small” that died during the nursery stage, no further plants died later in the experi- ment. In the field trial, no after effects of the Table 1. Effect ofrooting method and propagule size on weaning survival and plant size after a 4-week growing period in Experiment 4 with clone AF. Treatment Weaning Petiole Number survival, % length, cm of leaves Rooting method: direct rooting 86.7 a 6.9 a 4.9 a direct rooting + 83.3 a 5.6 a 4.3 a Floramon A in vitro rooting 88.9 a 8.0 a 4.7 a Propagule size: small 80.0 a 4.2 c 4.1 b medium 87.8 a 6.5 b 4.4 b large 91.1 a 9JU 5.2 a Values for rooting method and propagule size separately followed by the same letter are not signifi- cantly different at P = 0.05. 191 Research Note Research Note Table 2. Effect of rooting method and propagule size on plant size after a 7-week growing period in Experiment 4 with clone AF. Petiole Number Foliage fresh Foliage dry length, cm of leaves weight, g weight, g Rooting method: direct rooting 14.0 a 7.4 a 16.6 a 0.88 a direct rooting + 12.4 a 6.6 a 11.9 a 0.68 a Floramon A in vitro rooting 14.8 a 7.1 a 19.0 a 0.96 a Propagule size: small 11.2 c 6.3 b 6.0 c 0.31 c medium 13.3 b 7.2 a 13.6 b 0.79 b large 16.6 a 7.7 a 28.0 a 1.41 a Values for rooting method and propagule size separately followed by the same letter are not signifi- cantly different at P = 0.05. Table 3. Effect of propagule size on the further development and yield of clone AF during 1990-1992. Year and Variable date Propagule size small CV% medium CV% large CV% 1990 21 June Number of leaves 8 Aug. 3.3 39 3.1 32 15.3 35 3.3 24 15.6 3913.2 40 1991 23 May Number of leaves 24.2 16 26.8 23 26.7 23 Longest petiole, cm 24.4 15 24.4 20 24.8 13 1992 x) 22 May Petiole - length, cm - weight, g 34 9 18 34 5 108 10 35 4 100 14100 4 June Petiole - length, cm - weight, g 45 9 13 45 5 122 14 46 5 128 15121 No significant differences at P = 0.05. CV = coefficient of variation for respective variable in CV%. x) Five largest leaves per plant were harvested on both dates. rooting method were found (data not presented). The differences between propagule size catego- ries were not significant (Table 3). Variation in the measured growth parameters between indi- viduals was considerable. Among the 47 surviv- ing plants, no morphologically aberrant ones were found. Discussion The nutrient medium gave an acceptable multi- plication rate for clone AF. However, for cv. Vic- toria, the medium did not seem optimal for mi- cropropagation because of vitrification and the low multiplication rate in Experiment 2. The dif- 192 Agricultural Science in Finland 3 (1994) Research Note ference in multiplication rate might be due to genotype since cv. Victoria propagates slowly in vivo (Walkey and Matthews 1979). In Experi- ment 3, the multiplication rate of cv. Victoria was increased by using propagules from non- sprouting instead of sprouting buds. The results of Experiments 2 and 3 are, however, not directly comparable since the time of year for bud exci- sion can affect their hormone and carbohydrate levels (George and Sherrington 1984). The high callus occurrence especially in cv. Victoria indicates a too high auxin level, which agrees with the findings of Camara Machado et al. (1990) with cv. Holsteiner Blut. The occa- sionally high incubation temperature in the mul- tiplication stage possibly enhanced callus forma- tion and reduced the multiplication rate since the efficiency of cytokinins is found to decrease as the temperature rises, but that of auxins to in- crease (George and Sherrington 1984).Further, less callus formation and a higher multiplication rate have been obtained in rhubarb by using glu- cose instead of sucrose as carbohydrate source (Rumpunen 1990). In a preliminary test with rhubarb, direct root- ed shoots survived well but grew more slowly than in vitro rooted ones (Rumpunen 1990). In our study with clone AF, direct and in vitro root- ing gave nearly identical results. The small size of the propagules was no obstacle to good sur- vival, but 7 weeks after transplanting, plants from small propagules were clearly inferior in weight to plants from larger propagules. They also had fewer leaves but, at the nursery stage, the number of leaves was almost the same in all propagule categories. The risk of genetic changes is minimal if shoot tip explants and optimum nutrient medium are used (George and Sherrington 1984). In our field trial, no morphologically aberrant plants were observed but, between individuals, the variations in number of leaves and petiole length were con- siderable. These variations could be due to envi- ronmental factors. Somaclonal variation cannot be excluded, but since the variations decreased with time, they would most likely be of epigenet- ic character, possibly associated with virus elimi- nation. In conclusion, micropropagated shoots of clone AF can be rooted directly, and even shoots 1.5 to 2 cm in height do root and develop well. For cv. Victoria, a suitable initiation and multiplication medium should be designed. References Camara Machado, M. L. da, Camara Machado, A. da, Hanzer, V., Kalthoff, 8., Weiss, H., Mattano- vich, D., Regner, F. & Katinger, H. 1990. In-vitro Vermehrung von Rhabarber {Rheumrhaponticum). Mit- teilungen Klostemeuburg, Rebe und Wein, Obstbau und Friichteverwertung 40: 84-87. Capellades, M., Vanderschaeghe, A., Lemeur, R. & Debergh, P. 1990.How important is photosynthesis in micropropagation? Current Plant Science and Biotech- nology in Agriculture 8: 29-38. Dordrecht. Case, M. W. 1970. Production and propagation of virus free stocks of rhubarb. Ministry ofAgriculture, Fisher- ies and Food. Experimental Husbandry Farms and Ex- perimental Horticultural Stations. Annual Report 11: 133-134. Conner, A. J. & Thomas, M. B. 1981. Re-establishing plantlets from tissue culture: a review. Combined Pro- ceedings of the International Plant Propagators’ Socie- ty 31: 342-257. Debergh, P. C. & Maene, L. J. 1981.A scheme for com- mercial propagation of ornamental plants by tissue cul- ture. Scientia Horticulturae 14: 335-345. George, E. F. & Sherrington,P. D. 1984. Plant propaga- tion by tissue culture. Handbook and directory of com- mercial laboratories. 709 p. Basingstoke. Murashige, T. & Skoog, F. 1962. A revised medium for rapid growth and bioassays with tobacco cultures. Phys- iologia Plantarum 15: 473-497. Norman, B. 1978. Forced rhubarb revival urged by Stock- bridge House EHS. Grower 89: 904, 906. Pierik, R. L. M., Banga, M. & Janson, J. 1989. Vegeta- tieve vermeerdering van rabarber in kweekbuizen. [Veg- etative propagation of rhubarb in growing tubes.] Pro- phyta 43: 13-14. Roggemans, J. & Claes, M.-C. 1979. Rapid clonal pro- pagation by in vitro culture of shoot-tips. Scientia Hor- ticulturae II; 241-246. Rumpunen, K, 1990. Mikroförökning av rabarber. Sum- 193 Agricultural Science in Finland 3 (1994) Research Note mary: Micropropagation of rhubarb. Sveriges Lant- bruksuniversitet. Avdelningen för Hortikulturell Växt- förädling. Balsgärd - Verksamhetsberättelse 1988-1989: 126-133. Walkey, D. G. A. 1968. The production of virus-free rhubarb by apical tip culture. The Journal of Horticul- tural Science 43: 283-287. - & Matthews, K. A. 1979. Rapid clonal propagation of rhubarb (Rheum rhaponticum L.) from meristem-tips in tissue culture. Plant Science Letters 14: 287-290. Wardle, K., Dalsou, V, Simpkins, I. & Short, K. C. 1983. Re-distribution of rubidium in plants of Chry- santhemum morifolium Ram. cv. Snowdon derived from tissue cultures and transferred to soil. Annals of Bota- ny 51: 261-264. Manuscript received October 1993 SELOSTUS Raparperin mikrolisäys, versojen juurtuminen karaisuvaiheessa ja myöhempi kasvu CarolaLassus ja Irma Voipio Helsingin yliopisto Tutkimuksessa haluttiin selvittää, onnistuuko mikrolisä- tyn raparperin suora juurrutus turve-vermikuliittialustalle, onko eri pituisten versojen juurtumisessa eroja ja ilme- neekö myöhemmässä kasvussa juurrutustavasta tai lisäyk- sessä käytetyn verson koosta johtuvia eroja. Lajiketta Victoria ja kloonia AF mikrolisättiin juurakon silmujen kärkisolukoista Murashigen ja Skoogin kehittä- mällä alustalla (MS-alusta), joka sisälsi 20 mg/1 sakka- roosia ja 8 mg/1 agaria. Aloitus-ja monistusvaiheissa alus- taan lisättiin 1 mg bentsyyliaminopuriinia ja 1 mg indoli- voihappoa litraa kohti. Juurrutusmenetelmän ja verson koon vaikutusta juurtumiseen ja myöhempään kasvuun tutkit- tiin käyttäen kloonia AF. Tämän kokeen taimien jatkokas- vua avomaalla seurattiin kolmen vuoden ajan. Klooni AF tuotti keskimäärin 3,2 versoa 4 viikossa. Lajikkeella Victoria tulos oli heikompi, ja kallusmuodos- tusta ja vesisoluisuutta esiintyi runsaasti. Victoria lajik- keen versojen tuotto parantui, kun lisäys tapahtui levossa olevista silmuista. Suoraan turve-vermikuliittialustalle is- tutetut kloonin AF versot juurtuivat yhtä hyvin (87 %) kuin ne, joiden istutusta edelsi juurrutusvaihe MS-alustal- la (89 %). Pienet versot (pisimmän lehden korkeus 1,5 - 2 cm) juurtuivat lähes yhtä hyvin kuin keskikokoiset (2,1 - 5 cm) tai suuret (5,1 - 10 cm). Suurimmista versoista kehittyneet taimet olivat karaisuvaiheen päättyessä kook- kaimmat. Taimisto-ja peltoviljelyvaiheissa ei havaittu juur- rutustavasta tai verson koosta johtuvia eroja. 2194 Agricultural Science in Finland 3 (1994)