ASPECTS REGARDING THE IN VITRO PROPAGATION OF HIGHBUSH BLUEBERRY CULTIVAR BLUE CROP

Bulletin UASVM, Horticulture 65(1)/8 pissn 183-55; eissn 183-539 ASPECTS REGARDING THE IN VITRO PROPAGATION OF HIGHBUSH BLUEBERRY CULTIVAR BLUE CROP Alexandru FIRA, Doina CLAPA, C. BADESCU Fruit Research Station Cluj, 5 Horticultorilor Street, 57, Cluj-Napoca, Romania, alexfiracluj@yahoo.com Keywords: blueberry, in vitro, Zeatin, -Isopentenyladenine, Thidiazuron, coconut water Abstract: The scope of this paper is to present the in vitro propagation of the highbush blueberry cultivar Blue Crop, a cultivar that has important difficulties regarding in vitro propagation as well as propagation by cuttings. As basal medium, Woody Plant Medium (WPM, after Lloyd & McCown) was used. As growth regulators, -Isopentenyladenine, Zeatin, Thidiazuron and raw coconut water obtained from ripe fruit were used. The iron source is also very important, FeNaEDDHA (Sequestrene 138) providing far better results than FeNaEDTA. FeNaEDDHA can either be added as iron supplement to the prepackaged media or it can be used as the only iron source in the media prepared from stock solutions of macroelements and microelements. The ph recommended for the media used for bluecrop in vitro propagation is 5. Also, some peculiarities of the in vitro propagation of cultivar Blue Crop are presented, in comparison with other cultivars. INTRODUCTION The blueberry cultivar Blue Crop was obtained in the USA by hybridizing the cultivars (Jersey x Pioneer) x (Stanley x June) and introduced into culture in 195. It is a vigorous, very productive cultivar, resistant to frost and drought, with very good quality fruit, easy to grow, but relatively difficult to propagate, the cuttings being dificult to root. In vitro propagation of cultivar Blue Crop also presents important difficulties, multiplication rate being relatively low. Its capacity for the in vitro neogenesis of shoots is also low. The highbush blueberry is propagated in vitro on an industrial scale in the USA as well as in some in vitro culture laboratories in Europe. Among the authors that have done research for the in vitro propagation of this species we cite: Chandler C.K., Draper A.D., Eccher T., Noe N., Piagnani C., Castelli S. etc. (1,, 3,, 5, 6, 7, 8, 9,1, 11, 1, 13). In the year, at the Fruit Research Station in Cluj, research was begun for the in vitro propagation of the highbush blueberry. MATERIALS AND METHODS In order to establish the optimal variant of media for this cultivar several experiments were done by using Woody Plant Medium as basal medium (after Lloyd and McCown), either original or modified and, as growth regulators, -Isopentenyladenine, Zeatin, Thidiazuron and coconut water deriving from ripe fruit were used at various concentrations (Table 1). Vitamin C (ascorbic acid) was used in powder form. The ph of the media was adjusted to 5. As gelling agents, Isubgol and agar were used. Stock-solutions of vitamins B1, B6 and nicotinic acid were used as well as stock solutions of hormones at. Myo-inositol was added to the media as powder. All the ingredients were added to the media before autoclavation, even 1

Vitamin C, a substance that easily decays at high temperatures. Coconut water was used at 1 ml/l of nutritive medium. Culture initiation was done in test tubes on WPM + 5 mg/l -Ip. For multiplication, Magenta GA7 polycarbonate vessels were used, with polypropylene caps. The cultures were incubated in the growth chamber in artificial light provided by fluorescent tubes. Light intensity was of about lux and temperature was of -6 C. For multiplication, 1-1.5 cm long shoot fragments resulted from in vitro cultures were used as plant material. The cultures were watched for two months in the case of using Isubgol, for 7 weeks when using Zeatin and for three months in the experiments with Zeatin, coconut water and the ones with -Ip and vitamin C, because these variants provided slower growth. Table 1 Modified Woody Plant Medium, experimental variants Component WPM * salts Sequestrene 138 FeNaEDTA Myo-inositol Vitamin B 1 Vitamin B 6 Vitamin C Nicotinic acid Ip Zeatina TDZ Coconut water Agar Isubgol Sugar ph=5 *Woody Plant Medium Concentration Standard 1 mg/l 1 mg/l mg/l, 1, mg/l 5 mg/l, 3 mg/l.1,.,.5 mg/l 1 ml/l 5 g/l 15 g/l 3 g/l RESULTS AND DISCUSSIONS Cultivar Blue Crop is difficult to grow in vitro on the culture medium currently used (Table ) for the micropropagation of the highbush blueberry. On this medium short, thin and chlorotic shoots were obtained, and their bases get necrosed in 3-5 weeks after inoculation. Table Woody Plant Medium modified Component WPM salts FeNaEDTA Myo-inositol Vitamin B 1 Vitamin B 6 Nicotinic acid -Isopenteniladenine Sugar Agar ph=5 Concentration.3 g/l 1 mg/l mg/l 5 mg/l 3 g/l 5 g/l 15

As a measure of emergency the plants were passed onto a modified culture medium, in which agar was replaced with Isubgol and FeNaEDTA with Sequestrene 138 (Table 3). Variants of media with Sequestrene 138 and Isubgol for blueberry cultivar Blue Crop Table 3 Variants V V 1 V Basal medium+ growth regulators WPM* WPM* WPM* Iron supplement FeNaEDTA FeNaEDTA Sequestrene 138 1 mg/l Gelling agent Agar 5 g/l Isubgol 15 g/l Isubgol 15 g/l *According to Table The multiplication rate of the blueberry on the agar-gelled medium was of 1.3 and for the two variants with Isubgol the multiplication rates were 3.9 for variant V1 and. for variant V (Figure 1) Multiplication rate in cultivar Blue Crop.5 3.5 3.5 1.5 1.5 V V1 V Experimental variants Multiplication rate Multiplication rate in cultivar Blue Crop on media gelled with Isubgol Figure 1 The results demonstrate that for cultivar Blue Crop the product Isubgol used as a gelling agent is more adequate that the agar used initially, greatly increasing multiplication rate, shoot length and thus the quality of the plant material resulted from in vitro culture. The compound Sequestrene 138 also has a beneficial effect in the in vitro culture of the blueberry, the plants being greener, with a healthier appearance. Another measure taken for improving the in vitro propagation of cultivar Blue Crop was the replacement of -Ip with Zeatin. The results obtained are presented in Table 3 in comparison with other blueberry cultivars that were studied. Seven weeks after inoculation two vessels were taken randomly from each cultivar and the multiplication rate was the following (Table ): In cultivar Blue Crop the average number of shoots/plantlet was 7.3. Although the multiplication rate in cultivar Blue Crop (8.5) was lower in comparison with the other cultivars, it is significantly higher than on the best of the preceding variants (.) and the plantlets are more vigorous, do not get necrosed and can be successfully acclimatized. Another growth regulator tested for the in vitro propagation of cultivar Blue Crop was TDZ used in concentrations of.1,.,.5 and.. 16

Multiplication rate in cultivar Blue Crop on medium with Zeatin Cultivar Blue Crop Duke Elliott Hannah`s Choice No. of vessels used for multiplication No. of obtained vessels 17 33 3 1 Multiplication rate 8.5 16.5 16 1.5 Table It has been found that TDZ provides slow growth and relatively low multiplication rate, the resulting plants being deformed, with large leaves, with callus and compact masses of buds at the base of the stem. TDZ could be used for keeping cultivar Blue Crop in vitro for a long period of time. The average no. of shoots/plantlet obtained on the variants with TDZ in cultivar Blue Crop in comparison with other cultivars is presented in Table and Figure. As a growth regulator for the in vitro multiplication of cultivar Blue Crop raw coconut water was also used, in the concentration of 1 ml/l. On this medium the Blue Crop plantlets grew slowly and a small number of short shoots resulted. The average no. of shoots/plantlet is presented in Figure 3. under the influence of various concentrations of TDZ 8 6 Blue Crop Duke Elliott.. mg/l.5 mg/l. 5 3 1 resulted under the influence of coconut water 1.91 3.5.5 Blue Crop Duke Elliott Average no. of shoots/plantl et Figure Figure 3 The effect of coconut water upon blueberry shoot proliferation Another aspect to be studied was the effect of vitamin C upon the in vitro multiplication of this blueberry cultivar. For this purpose, 3 variants of media were tested using the multiplication medium from Table 3 to which Vitamin C was added in 3 concentrations: V1- mg/l, V 1 mg/l and V3 mg/l. Vitamin C was added to the media before autoclavation. The reaction of cultivar Blue Crop on these media was compared with that of variants Duke and Elliott, and the results are presented in Table 5. We mention that in cultivar Blue Crop the largest number of shoots/vessel was obtained at the concentration of mg/l Vitamin C. The presents of Vitamin C also prevented the bases of the plantlets from blackening and the plants were relatively vigorous, suitable for acclimatization. By comparing the four variants tested for the in vitro multiplication of the highbush blueberry cultivar Blue Crop, respectively the variants with Zeatin, coconut water, TDZ and 17

-Ip + Vitamin C (Table 6 and Fig. ) there results that the best variant is the one with mg/l Zeatin. Table 5 The effect of Vitamin C upon blueberry in vitro multiplication Cultivar Variant Average no. of shoots/vessel Blue Crop Average no. of shoots/plantlet 1 116 7.5 68.5 Duke Elliott 3 7.37 1 86 5.37 53 3.31 3 69.3 1 81 5.6 19 9.31 3 15.5 9.53 obtained on the four variants tested Table 6 Variant V 1 Coconut water 1 ml/l 1.91 V Thidiazuron, mg/l 5.9 V 3 - -Ip (5 mg/l ) + vitamin C ( mg/l) 7.5 V - mg/l zeatin 7.3 Average number of shoots/plantlet in the different experimental variants. 8 7.5 7.3 6 5.9 Average no. of shoots/plantlet 1.91 V1 V V3 V Experimental variants Average number of shoots/plantlet in cultivar Blue Crop on various experimental variants Figure On the variant with Vitamin C almost the same number of shoots/plantlet were obtained, but the shoots are shorter, up to 3- cm as compared to the ones on Zeatin which have up to 7-8 cm, thus offering the largest multiplication rate. 18

CONCLUSIONS The micropropagation of the highbush blueberry cultivar Blue Crop is relatively difficult. The culture medium recommended for the micropropagation of cultivar Blue Crop is modified WPM (Table ) with maximum mg/l Zeatin as growth regulator. The use of Zeatin strongly stimulates shoot growth and considerably increases multiplication rate in the highbush blueberry. Culture on media with 5 mg/l -Ip and mg/l Vitamin C is also recommended, as it confers greater vigor to the shoots. It is not recommended to use TDZ as growth regulator, as it provides slower growth and lower multiplication rates, the resulting plants also being deformed. However, it is possible to maintain cultivar Blue Crop on the media with TDZ for long periods of time. It is also possible to use raw coconut water but a low number of shoots is obtained and the plantlets grow slowly. Isubgol can be successfully used as a gelling agent in blueberry in vitro culture, especially for cultivar Blue Crop, strongly stimulating plant growth. The compound Sequestrene 138 as iron source gives better results than FeNaEDTA in the blueberry in vitro cultures, conferring dark green colour to the plantlets. REFERENCES 1. Chandler, C. K., A. D. Draper, 1986, Effect of zeatin and ip on shoot proliferation of three blueberry clones in vitro, Hort Science 1: 165-166.. Da Silva, L. C., M. W Schuch, J. A. De Souza., A. C. Erig., L. E. C. Antunes, 6, Nutritive medium, growth regulators and cold in the in vitro establishment of blueberry (Vaccinium ashei Reade) Cv. Delite, R. Bras, Agrociência, Pelotas, v. 1, n., p. 5-8. 3. Eccher, T., N. Noe, 1989, Comparison between ip and zeatin in the micropropagation of highbush blueberry (Vaccinium corymbosum), Acta Horticulturae. Fourth international symposium on Vaccinium culture, East Lansing, Michigan, USA, 13-17 Aug. 1988, 1: 185-19.. Eccher, T., N. Noe, C. Piagnani, S. Castelli, 1986, Effects of Increasing Concentration of BAP and ip on in vitro culture of Vaccinium corymbosum, Acta Horticulturae 179, II : 879-881. 5. Gonzalez, M. V., M. Lopez, A. E. Valdes, R. J. Ordas,, Micropropagation of three berry fruit species using nodal segments from field-grow plants, Annals of Applied Biology, vol. 137, no.1, pp. 73-78. 6. Gajdošová, A., M. G. Ostrolucká, G. Libiaková, E. Ondrušková, D. Šimala, 6, Microclonal propagation of Vaccinium sp. And Rubus sp. and detection of genetic variability in culture in vitro, Journal of Fruit and Ornamental Plant Research Vol. 1 (Suppl. 1). 7. George, E. F., 1993-1996, Plant Propagation by Tissue Culture, Part, Exegetics Limited, Edington, Wilts. 8. Jain, R., S. B. Babbar, 5. Guar gum and isubgol as cost-effective alternative gelling agents for in vitro multiplication of an orchid, Dendrobium chrysotoxum, Current Science, vol. 88, no., 5 January. 9. Krzewynska, D et al. Growth and yielding of highbush blueberry (Vaccinium corymbosum L. ) propagated by traditional and in vitro methods. Zeszyty naukowe Instytutu Sadownictwa I Kwiaciarstwa, Tom 1. 1. Marcotrigiano, M., S. P. McGlew, 1991, A Two-stage Micropropagation System for Cranberries, J. Amer. Soc. Hort. Sci. 116(5):911-916. 11. Orlikowska, T., 1986, Micropropagation of Highbush Blueberry, Fruit Science Reports, Vol. XIII, No. 3 1. Ostrolucká M. G. et a.l, In vitro propagation of Vaccinium species, Acta Universitatis Latviensis, Biology, Vol. 676. pp.7-1. 13. Zimmerman, R. H., O. C. Broome, 198, Blueberry micropropagation, USDA-SEA Agr. Res. Results ARRNE. 11, -7. 19