Butternut (Juglans cinerea L.), a native hardwood to the northeastern United States, is a valuable species for its wood and edible nuts. Butternut is becoming endangered in its native range as a result of a virulent fungal (perennial canker) pathogen, Sirococcus clavigignenti - juglandacearum. Micropropagation techniques are being developed to clone disease-resistant specimens. Axillary buds, obtained from 2-3-month old seedlings, were induced to break buds in vitro and form a single shoot when cultured on Murashige and Skoog (MS) medium supplemented with 200 mg/l casein hydrolysate, 3% sucrose, and 2 mg/l 6-benzylaminopurine. Roots were initiated on microshoots when cultured on half-strength MS medium containing 100 mg/l casein hydrolysate, 1.5% sucrose, and 0.5 mg/l indole-3-butyric acid for seven days in the dark. Adventitious roots elongated when shoots were placed in the light on the same medium, but with 2% sucrose, and no growth regulators. Rooted plantlets were successfully acclimated ex vitro. These results provide a basis for the development of techniques to micropropagate selected, mature, disease-resistant butternut germ plasm.
Paula M. Pijut
Paula M. Pijut and Melanie J. Barker
Butternut trees are becoming endangered as a result of butternut canker disease; thus, it is desirable to propagate disease-resistant trees for screening and provenance tests. The objective of this study was to determine the conditions necessary for successful cutting propagation. In 1998, 10 trees were selected from a 4-year-old butternut plantation located in Rosemount, Minn. Hardwood cuttings were collected 30 Mar., 21 Apr., and 6 May. The auxins, indole-3-butyric acid-potassium salt (KIBA) in water at 0, 29, or 74 mmol·L-1 were tested for root induction. The basal end of cuttings were dipped in treatment solutions for 10 to 15 s and placed in a 1 peat: 1 perlite mixture in Deepots™ (D40) in a mist bed. Mist was applied for 5 s every 15 min. Greenhouse conditions were: 12-h photoperiod provided by high-pressure sodium lamps (60 μmol·m-2·s-1), 22 °C, and bottom heat of 27 °C (heating pads). Softwood cuttings were collected 20 May, 18 June, 30 June, and 23 July. Rooting treatment solutions and greenhouse conditions were the same as for the hardwood cuttings, except no heating pads were used. Rooted cuttings were planted in Treepots™ (10 × 10 × 36 cm) and gradually hardened off from the mist bed. Hardwood cuttings from the first two collection dates did not initiate roots. Best rooting (12.5%) was achieved on hardwood cuttings collected 6 May using 29 mmol·L-1 KIBA. Softwood cuttings rooted to some degree at all concentrations of rooting solution and at every collection date. The greatest rooting (70%) was achieved using 74 mmol·L-1 IBA. In general, best rooting percentages were achieved with softwood cuttings collected 18 June and 23 July and treated with 62 mmol·L-1 KIBA or 74 mmol·L-1 IBA. Both rooted hardwood and softwood cuttings were successfully acclimatized from the mist bed and many have initiated new growth.
Rochelle R. Beasley and Paula M. Pijut
Fraxinus nigra Marsh. (black ash) is a native North American hardwood tree species that is ecologically important and has ethnobotanical significance to American Indian communities of the eastern United States. Black ash has immature embryos at seed set, combined with complex stratification requirements, making natural regeneration difficult. This, combined with the threat and devastation being caused by the emerald ash borer, makes an in vitro adventitious shoot regeneration and rooting protocol imperative for mass propagation, conservation, and genetic improvement of this species. Hypocotyls were cultured for 4 weeks on a modified Murashige and Skoog (MS) medium containing 13.3 μM 6-benzylaminopurine (BA) and 4.5 μM thidiazuron for adventitious shoot induction. Shoots continued to regenerate when explants were then cultured on MS medium supplemented with Gamborg B5 vitamins plus 0.2 g·L−1 glycine (B5G), 6.7 μM BA, 1 μM indole-3-butryic acid (IBA), and 0.29 μM gibberellic acid (GA3) for 4 weeks, followed by transfer to MSB5G medium with 13.3 μM BA, 1 μM IBA, and 0.29 μM GA3 for shoot elongation. Once elongated, the microshoots were continuously micropropagated through nodal sectioning, and cultured on MSB5G medium supplemented with 13.3 μM BA, 1 μM IBA, 0.29 μM GA3, and 0.2 g·L−1 casein hydrolysate. Rooting of elongated microshoots was successful using woody plant medium supplemented with 4.9 μM IBA and 5.7 μM indole-3-acetic acid with a 10-day initial dark culture for root induction followed by culture under a 16-h photoperiod. Rooted plantlets were successfully acclimatized to the greenhouse with 100% survival.
Paula M. Pijut and Melanie J. Moore
Juglans cinerea L. (butternut) is a hardwood species valued for its wood and edible nuts. Information on the vegetative propagation of this species is currently unavailable. Our objective was to determine the conditions necessary for successful stem-cutting propagation of butternut. In 1999 and 2000, 10 trees (each year) were randomly selected from a 5- and 6-year-old butternut plantation located in Rosemount, Minn. Hardwood stem cuttings were collected in March, April, and May. Softwood cuttings were collected in June and July. K-IBA at 0, 29, or 62 mm in water and IBA at 0, 34, or 74 mm in 70% ethanol were tested for root induction on cuttings. The basal end of cuttings were dipped in a treatment solution for 10 to 15 seconds, potted in a peat: perlite mixture, and placed in a mist bed for 5 to 8 weeks. Rooted cuttings were gradually hardened off from the mist bed, allowed to initiate new growth, over-wintered in a controlled cold-storage environment, and then outplanted to the field. For hardwood cuttings, rooting was greatest for those taken in mid-May (branches flushed out), 22% with 62 mm K-IBA and 28% with 74 mm IBA. Softwood cuttings rooted best when taken in June (current season's first flush of new growth or softwood growth 40 cm or greater) and treated with 62 mm K-IBA (77%) or 74 mm IBA (88%). For 1999, 31 out of 51 rooted softwood cuttings (60.8%) survived overwintering in cold storage and acclimatization to the field. For 2000, 173 out of 186 rooted softwood cuttings (93%) survived overwintering and acclimatization to the field. Chemical names used: indole-3-butyric acid-potassium salt (K-IBA); indole-3-butyric acid (IBA).
Kaitlin J. Palla, Rochelle R. Beasley, and Paula M. Pijut
The hard, strong, very close-grained wood of common persimmon (Diospyros virginiana L.; Ebenaceae) is desirable for specialty products such as golf club heads, percussion sticks, billiard cues, and for wood turnery. The edible fruit of cultivated varieties is sold as pulp for use in puddings, cookies, cakes, and custards. Persimmon is usually propagated by grafting. Own-rooted clonal persimmon could offer several advantages to specialty fruit growers such as elimination of grafting, graft incompatibility issues, and improved rootstocks for variety testing. Four mature, grafted (male and female) persimmon genotypes and one hybrid were used for nodal explant culture. Nodal stem explants were cultured on Murashige and Skoog (MS) medium containing 10 μM zeatin, 3% (w/v) sucrose, and 0.7% (w/v) Bacto agar. Explants were routinely transferred to fresh medium every 3 weeks until shoot cultures were established. All nodal explants excised from grafted greenhouse plants produced at least one viable shoot. For in vitro rooting of microshoots, half-strength MS medium with 0, 5, 10, or 15 μM indole-3-butyric acid (IBA), 0.1 g·L−1 phloroglucinol, 3% (w/v) sucrose, and 0.7% (w/v) Bacto agar were tested with a 10-day dark culture treatment followed by culture in the light. Best rooting (14% to 87%) was achieved on medium containing 5 μM IBA for the common persimmon genotypes with means averaging from 0.5 to 3.9 roots per shoot. Ninety-one percent rooting with 5.3 ± 2.6 roots per shoot was achieved for the hybrid persimmon. Rooted plants were successfully acclimatized to the greenhouse.
Ana Carolina Espinosa, Paula M. Pijut, and Charles H. Michler
A complete regeneration protocol was developed for Prunus serotina Ehrh., an important hardwood species for timber and sawlog production in the central and eastern United States. Nodal sections were cultured on Murashige and Skoog (MS) medium supplemented with 4.44 μm 6-benzylaminopurine (BA), 0.49 μm indole-3-butyric acid (IBA), and 0.29 μm gibberellic acid (GA3). In vitro leaf explants of three genotypes were placed on woody plant medium (WPM) supplemented with 0, 2.27, 4.54, or 6.81 μm thidiazuron (TDZ) in combination with 0, 0.54, 1.07, or 5.37 μm naphthaleneacetic acid (NAA), and on WPM supplemented with 0, 4.44, 8.88, or 13.32 μm BA in combination with 0, 0.54, 1.07, or 5.37 μm NAA. Cultures were maintained either in continuous darkness for 5 weeks, or in the dark for 3 weeks and then transferred to a 16-hour photoperiod. TDZ and the genotype had a significant effect on the number of shoots regenerated. The maximum mean number of shoots regenerated per explant (5.05 ± 1.14) was obtained with 2.27 μm TDZ plus 0.54 μm NAA with the 3-week dark period then light treatment. The highest percent shoot regeneration (38.3) and mean number of shoots (4.13 ± 0.97) was obtained with 6.81 μm TDZ plus 1.07 μm NAA. The highest rooting (27%) of adventitious shoots and number of roots per shoot (2.3 ± 0.2) was obtained with 2.5 μm IBA when shoots were maintained for 7 days in the dark on rooting medium before transfer to a 16-hour photoperiod. The highest rooting (70%) of nodal explant-derived stock cultures and number of roots per shoot (2.7 ± 0.9) was also obtained with 2.5 μm IBA, but when shoots were maintained for 4 days in the dark before transfer to a 16-hour photoperiod. In total, 86% of the plantlets survived acclimatization to the greenhouse and 100% survival after overwintering in cold-storage.