A New Method for Rapid In Vitro Propagation of Apple and Pear

Improved in vitro clonal propagation methods are valuable tools for nurseries and growers, and are essential for manipulation and improvement of tree fruit germplasm using the tools and techniques of biotechnology. We have developed a rapid shoot multiplication procedure for clonal propagation of apple, Malus ×domestica cv. Gale Gala and pear, Pyrus communis L. cv. Bartlett. Rapid clonal multiplication was achieved after the following series of steps: pre-conditioning of micropropagated shoots, sectioning pre-treated stems into thin slices, placing slices onto shoot induction medium and incubating directly under cool-white fluorescent lights or after a brief dark incubation. Multiple induction of shoots recovered from stem slice explants within three weeks of culture. A maximum of 37% of cultured apple stem slices, and 97% of pear stem slices, showed induction of shoots. More shoots were recovered on phytagel solidified shoot induction medium than on agar. Cultured stem slices of both apple and pear showed maximum recovery of shoots from shoot induction medium supplemented with thidiazuron (TDZ) compared to medium supplemented with BAP and kinetin. Under ideal conditions, pear stems generated four times the shoots as the same quantity or length of apple shoots. Micropropagated shoots were rooted and transferred to the greenhouse and field nursery for further evaluation. Chemical names used: N-phenylN′-1,2,3-thidiazol-5-ylurea (thidiazuron or TDZ); 6-benzylaminopurine (BAP). lary buds. Using this method, hundreds of plants were recovered from a single source stem within a short period of time. The study reported here describes results of an enhanced shoot multiplication system in both apple and pear. Materials and Methods Micropropagation of apple and pear. Micropropagation of apple (cv. Gale Gala) and pear (cv. Bartlett) was established using the buds of mature trees (Van Well Nursery, Wenatchee, Wash.). Cultures were maintained under cool-white fluorescent lights (38–40 μmol·m·s) in a growth chamber with 24 ± 2 °C and 16/8h photoperiod. Apple micropropagation medium (GM, Table 1) consisted of phytagel (0.25%) solidified medium with MS salts (Murashige and Skoog, 1962), B5 vitamins (Gamborg et al., 1968), glycine (26.6 μM), sucrose (87.6 mM), cefotaxime (209.4 to 418.8 μM), 6-benzylaminopurine (BAP, 4.4 μM), kinetin (13.9μM) and pH 5.8. Pear cultures were maintained in a phytagel solidified medium (BM, Table 1) with salts of Quoirin and Lepoivre (1977) (QL) salts, Staba vitamins (Staba, 1969), glycine (26.6 μM), sucrose (87.6 mM), cefotaxime (209.4 μM), BAP (2.2 μM) and kinetin (4.6 μM). Green shoots were separated and transferred to fresh medium every four weeks (transfer generation). Preconditioning. Preconditioning of shoots (i.e., source stems) from micropropagation cultures is a prerequisite step essential to increase the density of leaves (i.e. increase the number of axillary buds) on source stems. High leaf density was achieved by increasing the cytokinin composition in the micropropagation medium. Apple preconditioning medium was the same as apple propagation medium with BAP (4.4 or 8.8 μM) and kinetin (13.9 or 23.2 μM) and pear preconditioning medium was the same as pear micropropagation medium with BAP (13.2 μM) and kinetin (23.2 μM). Preconditioning treatment was carried out for two transfer generations under the growth conditions described earlier. Making stem slices and recovery of multiple shoots. Preconditioned shoots (1 to 2 cm long) with a high leaf density were selected for the stem slice procedure. Leaves were removed from preconditioned shoots and shoots were then sliced with a sterile doubleedged platinum razor blade. Multiple crosssectional slices <1.5 mm were made along the length of each source shoot. More than one slice must go through each leaf axis (or axillary meristem) to achieve multiple shoots from each slice. Fifteen to thirty stem slices from a 1.0to 2.0-cm-long preconditioned source stem were placed on shoot induction (or recovery) medium (see below) in a Petri plate (100 × 25 mm) and incubated under cool-white fluorescent lights as described above. If the total number of slices from a single source stem exceeded 30 then the slices were explanted onto two Petri plates. Two to three source stems were used in each treatConventional clonal propagation methods such as budding and grafting are used successfully by many nursery growers. While successful, these methods are slow, labor intensive and may require large amounts of land. Alternatively, successful in vitro clonal propagation methods are reported in apple and pear for commercial applications in the tree fruit industry. Over the last two decades, several research groups established in vitro conditions that were suitable for micropropagation of apple (Marin et al., 1993; Noiton et al., 1992; Skirvin et al., 1986; Webster and Jones, 1991; Welander, 1985; Yepes and Aldwinckle, 1994; Zimmerman, 1986) and pear (Viseur, 1987; Xiao-Shan and Mullins, 1984) cultivar and rootstock material. These methods hold promise for producing more plant material in a shorter period of time with less labor and at lower costs. These in vitro propagation methods yield an average of 4 to 6 shoots from a single source shoot over a period of 4 weeks (the transfer generation). However, it often requires several transfer generations to recover a sufficient number of clonal plants necessary for this method to be effective in commercial application. Varietal expansion of apple and to some extent pear expected to meet the demands of changing market and orchard needs throughout the world. Recent trends in the apple industry indicate that older cultivars such as ‘Delicious’ and ‘McIntosh’ are gradually becoming obsolete with the introduction of new improved cultivars (Barritt, 1999). Consumer preference drives the continuous production of new cultivars, and most consumer purchases are based on the varietal name (Barritt, 1999). Future projections for the apple industry also indicate that demand for new varieties will continue to grow through the turn of the century [U.S. Dept. of Agriculture (USDA) World Agricultural Outlook Board, 1999]. As new cultivars continue to replace older cultivars, a rapid in vitro vegetative propagation method offers an accelerated method of production of new tree fruit cultivars/ rootstocks that may be extremely valuable for commercial and private nursery growers. We have used in vitro propagated shoots to develop a rapid clonal multiplication procedure in apple (cv. Gale Gala) and pear (cv. Bartlett) by applying microsurgery to axil6505, p. 1102-1106 10/12/01, 11:40 AM 1102 1103 HORTSCIENCE, VOL. 36(6), OCTOBER 2001 ment and the procedure was repeated once with selected treatments, and subsequently, several times in the transformation experiments (unpublished data). Some cultures were incubated first in the dark for 2 weeks and then transferred to light conditions. Effects of plant growth regulators and gelling agents (phytagel or agar) were examined for rapid recovery of multiple shoots. Plant growth regulators and their concentrations were selected based on the earlier experiments (Table 1). Multiple shoots from each stem slice were separated and transferred to micropropagation medium, first to petri plates (15 to 20 shoots per petri plate) and then phytatrays (Sigma) (six to nine shoots per phytatray) and maintained as described earlier. The stem slice procedure was successfully used in transformation of apple and pear (unpublished data). Rooting of propagated shoots. Single shoots from established cultures were separated and placed onto rooting medium. Although several media were evaluated for induction of roots in both apple and pear, only results from media that showed maximum root induction are presented in this report. Apple rooting medium consisted of MS salts, B5 vitamins, glycine (26.6 μM), casein hydrolysate (25 mg·L), sucrose (87.6 mM), indole-3-acetic acid (IAA, 17.1 μM), and pH adjusted to 5.8 prior to autoclaving. Pear rooting medium consisted of QL salts, Staba vitamins, glycine (26.6 μM), sucrose (87.6 mM), casein hydrolysate (25 mg·L), indole butyric acid (IBA, 24.6 μM) and pH adjusted to 5.8 prior to autoclaving. Both media contained either phytagel (0.25%) or agar (0.8%). Five to six shoots were placed in each phytatray containing rooting medium. Cultures were incubated under light as described earlier for 6 to 8 weeks. Randomly selected plants were transferred to peat pots filled with greenhouse soil mix and incubated under light at room temperature for 2 weeks. Established plants were then transferred to the greenhouse and later transplanted to large size pots (7.6 L pot). Data on plant survival was collected after establishment in the greenhouse pots. Some of these plants (22 ‘Gale Gala’ and 23 ‘Bartlett’) were also transferred to the field (Van Well Nursery) in Summer 1999 for future evaluation. Collection and analysis of data. Shoots from each slice and shoots from each source stem were counted four to 6 weeks after stem slice culture. A randomized block experimental design was applied for single variance of analysis (treatments vs. shoots per slice) because of unequal numbers of stem slices in each treatment. Mean values for each treatment were calculated using Statgraphics software (Statgraphics Plus, v. 4.0; Manugistics, Rockville, Md.). One-way analysis of variance was carried out with Statgraphics software to identify differences among treatments using an F test at a 95% level of confidence (P ≤ 0.05) and results of multiple range test was used to separate homogeneous groups among treatments. Table 1. Media used for rapid shoot induction in apple (cv. Gale Gala) and pear (cv. Bartlett).