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Regeneration of Blueberry Cultivars Through Indirect Shoot Organogenesis

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Regeneration of Blueberry Cultivars Through Indirect Shoot Organogenesis HORTSCIENCE 53(7):1045–1049. 2018. https://doi.org/10.21273/HORTSCI13059-18 wilt disease incited by Ralstonia solanacea- rum occurred in seven farms in six counties of Florida (Norman et al., 2018). This is Regeneration of Blueberry Cultivars a newly discovered disease causing signifi- cant damage to blueberries. The pathogen is through Indirect Shoot Organogenesis easily spread in water, soil, or through in- 1 fected plant materials. Propagation through Dongliang Qiu and Xiangying Wei in vitro culture has been considered the most College of Horticulture, Fujian Agriculture and Forestry University, Fuzhou, effective method for a rapid increase of Fujian Province 350002, China disease-free propagules on a year-round basis (Chen and Henny, 2008; Murashige, 1974). Shufang Fan Blueberries have been micropropagated Jingchu University of Technology, College of Biological Engineering and via shoot culture (Fan et al., 2017; Frett and Institute of Plant Germplasm Resources Exploitation and Utilization, Smagula, 1983; Litwinczuk, 2013; Lyrene, Jingmen, Hubei Province 448000, China 1980, 1981; Ruzic et al., 2012; Tetsumura et al., 2008) and through shoot organogenesis Dawei Jian (Billings et al., 1988; Callow et al., 1989; Cao Jingmen Forestry Bureau, Jingmen, Hubei Province 448000, China and Hammerschlag, 2000; Debnath, 2009; Liu et al., 2010; Rowland and Ogden, 1992). Jianjun Chen1 Shoot culture is the proliferation of existing University of Florida, IFAS, Mid-Florida Research and Education Center, meristems, whereas shoot organogenesis re- fers to regeneration from explants without 2725 South Binion Road, Apopka, FL 32703 preexisting meristems. The latter generally Additional index words. callus, Ericaceae, micropropagation, shoot regeneration, Vaccinium, gives rise to a large number of shoots and is zeatin considered more efficient for plant multipli- cation. However, shoot organogenesis has Abstract. Leaf explants derived from in vitro–grown shoots of blueberry cultivars only been established in a limited number Bluejay, Pink Lemonade, Sunshine Blue, and Top Hat were cultured on woody plant of cultivars. Increasing evidence shows that medium (WPM) supplemented with 9.12 mM 6-(4-hydroxy-3-methylbut-2-enylamino) protocols developed for regenerating blue- purine or zeatin (ZT) in combination with 1.23, 2.46, or 4.92 mM indole-3-butyric acid berries are cultivar specific (Debnath, 2005, (IBA). Calluses were induced from the explants and adventitious shoots were regen- 2009; Liu et al., 2010; Meiners et al., 2007), erated. ‘Sunshine Blue’ and ‘Top Hat’ produced more than four shoots per explant but implying that different cultivars may require shoot numbers were less than one for each ‘Pink Lemonade’ explant and about 0.2 per different protocols for regeneration. Further- ‘Bluejay’ explant. The results indicate that there is significant difference among cultivars more, shoot organogenesis is one of the in indirect shoot organogenesis. The differences may be related to their diverse genetic regeneration pathways for genetic transfor- background as they are polyploid hybrids. Microcuttings derived from adventitious mation (Song and Sink, 2004). Thus far, few shoots of ‘Sunshine Blue’ rooted in vitro in WPM medium supplemented with 9.84 mM cultivars have been genetically transformed IBA and also rooted ex vitro in a peat-based substrate after cuttings were dipped or not (Gao et al., 2016; Song and Sink, 2004); the dipped in IBA solutions. Direct rooting of microcuttings in the peat-based substrate was lack of reliable regeneration systems could be effective, suggesting that in vitro rooting may not be necessarily needed. Survival rate of one of the limiting factors. ex vitro–rooted plants in a shaded greenhouse was high, more than 90%. The established In addition, rooting of microcuttings is an shoot regeneration protocols could be used for rapid propagation of ‘Sunshine Blue’ and important part of micropropagation. How- ‘Top Hat’ and for cultivar improvement through genetic transformation. ever, blueberry microcuttings are largely rooted ex vitro and little information is avail- able about in vitro rooting of blueberries Blueberries (Vaccinium spp.) are small- et al., 2016; Debnath, 2007). Among them, V. (Debnath, 2007; Litwinczuk, 2013). It is fruit crops and have become increasingly angustifolium, V. corymbosum, and V. myr- uncertain if rooted plantlets could have popular with consumers because of their tilloides are tetraploid with the chromosome higher survival rates after being transplanted high nutraceutical and pharmaceutical value number of 2n =4x = 48, whereas V. virgatum into soilless substrates. (Basu et al., 2010; Miller and Shukitt-Hale, is hexaploid with 2n =6x = 72 (Brevis et al., The present study was intended to de- 2012; Vendrame et al., 2016; Whyte and 2008). The highbush blueberries are by far velop protocols for regeneration of four Williams, 2015). There are five main groups the most important commercial crop. World- blueberry cultivars whose protocols for ad- of blueberries: 1) northern highbush (Vacci- wide highbush blueberry production in- ventitious shoot production have been not nium corymbosum L.); 2) southern highbush, creased from 58,400 ha in 2007 to 110,800 reported. As part of the effort, two of the developed from crosses between V. corym- ha in 2014 (Brazelton, 2015). North America four cultivars were successfully regenerated bosum, Vaccinium darrowii Camp, and other represented more than 50% of the production through indirect shoot organogenesis. Micro- blueberry species; 3) lowbush (Vaccinium area and accounted for almost 60% of the cuttings derived from adventitious shoots of angustifolium Ait., Vaccinium myrtilloides global highbush blueberry production in a cultivar were evaluated for both in vitro and Michx., and Vaccinium boreale Hall and 2014 (Brazelton, 2015), of which Florida ex vitro rooting. Results suggest that the Aald.); 4) half-high derived from crosses blueberry production acreage increased by developed protocols could be used for micro- between highbush and lowbush; and 5) rab- 73% from 2007 to 2012 (USDA, 2013). propagation, genetic transformation or both biteye (Vaccinium virgatum Ait.) (Caspersen Blueberries are traditionally propagated of the two cultivars. via stem cuttings (Marino et al., 2014), but not all cultivars can be effectively propagated Materials and Methods through this method (Lyrene, 1981). Stem Received for publication 9 Mar. 2018. Accepted cuttings are also not efficient in increasing Plant materials and culture medium. In for publication 18 May 2018. This work was supported by the Fujian Fumin numbers of starting materials or propagules vitro cultured four cultivars, representing Foundation and New Century Talent Training for commercial introduction of new culti- four of the five groups of blueberries, were Foundation of Fujian Province. vars (Miller et al., 2004). Furthermore, stem used in this study: Bluejay (V. corymbosum 1Corresponding authors. E-mail: qiudongliang@ cuttings may bring or spread pathogens L.) which is a northern highbush cultivar; aliyun.com or jjchen@ufl.edu. (Tzanetakis et al., 2017). Recently, a bacterial Pink Lemonade (a V. virgatum rabbiteye HORTSCIENCE VOL. 53(7) JULY 2018 1045 hybrid derivative); Sunshine Blue (V. cor- root numbers were recorded after 10 weeks of and fertigated with nitrogen at 100 mg·L–1 ymbosum L.), a southern highbush cultivar; culture. every 2 weeks. Survival rate and root num- and Top Hat, a half-high derived from a cross In vitro culture conditions. All culture bers per cutting were recorded 1 month later. between highbush and lowbush. experiments designed for evaluating ex- Experimental design and statistical analysis. Woody plant medium (Lloyd and McCown, plants, cultivars, and in vitro rooting were All experiments were arranged as a com- 1981) with vitamins (Product ID: L154; Phyto- maintained in a culture room under a 12-h pletely randomized design. Each culture ves- Technology, Shawnee Mission, KS) was used photoperiod provided by cool white fluores- sel (petri dish or glass baby food jar) was as a basal medium. After adding 3% sucrose cent lamps with a photon flux density of 50 considered an experimental unit for in vitro and 0.8% agar, the medium pH was adjusted to mmol·m–2·s–1 and temperature of 25 ± 2 °C. culture. For ex vitro rooting, six cell plugs 5.0 and autoclaved at 121 °C for 20 min. When Ex vitro rooting and acclimatization. were considered an experimental unit, and medium temperature dropped to 50 °C, Microcuttings of ‘Sunshine Blue’ were dip- each was repeated five times. Collected data filter-sterilized growth regulator solutions ped in deionized water, 4.92, 9.84, or 24.6 mM were subjected to ANOVA using SPSS 13.0 were added and mixed well before pouring IBA solutions for 10 s. Dipped cuttings were for Windows (SPSS, Chicago, IL). When into either glass baby food jars (150 mL) or rooted singly in cells of 72-plug trays filled significant differences (P < 0.05) occurred, petri dishes. with a peat-based substrate composed of means were separated using Duncan’s mul- The in vitro cultures were initially estab- 90% peat and 10% perlite with a pH of 5.0. tiple range test at P < 0.05 level. lished from stem explants cultured on WPM Meanwhile, plantlets derived from the in supplemented with 9.12 mM 6-(4-hydroxy-3- vitro rooting medium supplemented with Results methylbut-2-enylamino) purine or ZT and 9.84 mM IBA were transplanted into plug 0.025 mM IBAinbabyfoodjars.After trays filled with the same substrate. The
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