’ developed in Fukui Prefecture ( Sato et al., 2009 ). However, the efficiency of vegetative propagation by traditional methods is insufficient for mass propagation of plantlets. Adventitious shoots can be regenerated from cut surfaces of hypocotyls or stems
Takahiro Tezuka, Masashi Harada, Masahumi Johkan, Satoshi Yamasaki, Hideyuki Tanaka, and Masayuki Oda
L. Xu, G.F. Liu, and M.Z. Bao
inherited traits. In L. styraciflua , plants have been regenerated via adventitious shoots from mature leaf and petiole segments ( Brand and Lineberger, 1988 ) and hypocotyl segments ( Kim et al., 1997 ), and via somatic embryogenesis from hypocotyl
Xiaojuan Zong, Brandon J. Denler, Gharbia H. Danial, Yongjian Chang, and Guo-qing Song
significance was determined at 5% using the SPSS 20.0 program (IBM Corporation, Armonk, NY). Results Adventitious shoot regeneration. To evaluate the effects of exogenous hormones on adventitious shoot regeneration, 10 regeneration media (WPRM1–WPRM10) were
Sharon A. Bates, John E. Preece, and John H. Yopp
To increase adventitious shoot formation, we investigated the effects of the number of weeks on medium with high levels of plant growth regulators and seedcoat removal. Dissected white ash seeds were placed on a solidified MS medium containing 10 μM TDZ and 1 μM 2,4-D (shoot initiation medium). After 2, 3, or 4 weeks in vitro, explants were transferred to shoot elongation medium (3 μM TDZ, 1 μM BA, and 1 μM IBA). After 12 weeks, the greatest number (1.8) and longest shoots (18.7 mm) were in cultures incubated on the shoot formation medium for 3 weeks. In a separate experiment, dissected seeds were placed on shoot formation medium. Seedcoats were removed after 10 days in vitro. Explants were transferred to shoot elongation medium after 4 weeks in vitro. There were more shoots (2.5) on 12-week-old explants without seedcoats than on explants with seedcoats (0.9). This result may be related to inhibitors in the testa.
Karim H. Al-Juboory and Jabar H. Al-Niami
Leaves of wild apple (Malus domestica Borkh) were excised from in vitro grown shoots transversely cut into halves and plated onto petri dishes containing regeneration media. Cultures were kept in the dark for three weeks before adventitious shoots were observed. Callus from leaf explants produced adventitious shoots after 3 months of in vitro culture. Callus were cultured on Nitsch and Nitsch medium supplemented with a range of BA (0.0–2.0 μm) and NAA (0.0–10 μm). BA at 10 μm combined with NAA (0.5 μm) proved most effective for stimulating shoot proliferation of cultured apple. Plantlets from tissue culture were easily transferred to the greenhouse environment.
Giovanni Iapichino, Tony H.H. Chen, and Leslie H. Fuchigami
An efficient adventitious shoot production protocol has been developed for Rhododendron laetum × aurigeranum. Shoot tips taken from greenhouse-grown plants were cultured on Anderson's medium supplemented with 74 μM 2iP. Axillary shoots were excised and cultured on medium containing 23 μM IAA and 74 μM 2iP. After 6 months, brown callus developed at the cut surfaces of the shoot-tip explants. This callus produced many adventitious shoots (up to 70 per explant). Clusters of adventitious shoots were divided, subculture, and continued to proliferate shoots. An estimated 1600-fold increase in the number of shoots could be readily achieved in 6 months. In vitro rooting of adventitious shoots was accomplished in 4 weeks. Seventy-three percent of shoots rooted on 1/4 strength Anderson's medium supplemented with 28 μm IAA. Plantlet survival was 100%3 weeks after transfer to soil. Chemical names used: 1-H-indole-3-acetic acid (MA); N-(3 -methy1-2-butenyl) -1H-purine-6 amine (2iP).
María Victoria González, Manuel Rey, Raffaela Tavazza, Stefano La Malfa, Luigi Cuozzo, and Giorgio Ancora
Plant regeneration was obtained from adventitious buds induced in isolated cotyledons of Italian stone pine (Pinus pinea L.). The best results for bud induction were obtained by using half-strength LePoivre medium with 4.5 μM 6-benzyladenine for 30 days. Shoot elongation was achieved in the same medium without growth regulators but with the addition of 0.5% activated charcoal. The induction medium was the best also for shoot multiplication, but it was necessary to include subcultures on elongation medium. The slow elongation rate of adventitious shoots remains the greatest obstacle to multiplication. Root formation (15%) after 5 months was observed when shoots were cultured on elongation medium for long periods.
Sharon Bates, John E. Preece, John YOPP, and Robert Trigiano
Dissected white ash seeds were placed on a MS basal medium containing 10 μM TDZ and 1 μM 2,4-D. Adventitious buds formed directly and indirectly on cotyledons and hypocotyls that were in contact with the medium. Histological observations after 7 days from initiation indicated early divisional events originated directly in subepidermal layers on adaxial portions of the cotyledons. As these cells divided, the growth ruptured the epidermis. Bud-like structures were seen at 3 weeks. After transfer to a secondary medium containing 3 μM TDZ, 1 μM BA, and 1 μM IBA, some of adventitious buds elongated. Efforts (gibberellin, etiolation, ABA, and silver nitrate treatments) to increase the number of elongated buds have been unsuccessful. Excised adventitious shoots were rooted under mist and established in the greenhouse.
Alan G. Smith and Elizabeth S. Zimmermann
Euonymus alata is an attractive landscape plant that has been reported to be an invasive species. Genetic modification through transformation is a method of reducing its invasiveness by producing sterile cultivars having limited or no seed production. A critical step in Agrobacterium-mediated gene transfer is the production of adventitious shoots. E. alata internodes and leaves from in vitro cultures were tested for adventitious shoot production on 16 plant growth regulator combinations: four levels of 6-benzylamino purine (BA) and three auxin treatments [0.5 or 0.25 mg·L-1 indole-3-butyric acid and 0.1 mg·L-1 naphthaleneacetic acid (NAA)], as well as no auxin. The optimal BA levels were found to be 0.5 or 1.0 mg·L-1 for maximizing the number of explants forming shoots and for producing the greatest number of shoots per explant. Culturing on NAA gave the greatest number of shoots per explant with both 0.5 and 1.0 mg·L-1 BA. Shoot production from internode segments was markedly superior to leaves. An initial dark treatment of 10 days did not influence shoot production. Using 1.0 mg BA with 0.1 mg·L-1 NAA, E. alata internodes were transformed with A. tumefaciens EHA105 carrying Kanamycin resistance and β-glucuronidase genes. Transformed shoots were selected on 30 mg·L-1 Kanamycin. Of the 36 shoots produced, 16 were confirmed to be transformed by β-glucuronidase histochemistry. Treatment with rooting powder containing indole-3-butyric acid did not aid rooting of shoots, but after 3 months in soil in high humidity, 21 of 24 E. alata shoots from tissue culture were rooted and acclimated.
Kenneth R. Schroeder and Dennis P. Stimart
One-centimeter hypocotyl explants from 2-week-old Antirrhinum majus L. (snapdragon) seedlings germinated and grown in vitro under 12-h cool-white fluorescent light and 12 h dark or 24 h dark were placed on Murashige and Skoog (MS) medium containing 0, 0.44, 2.22, 4.44, 8.88, or 44.4 μM N6-benzyladenine (BA). Cultures were maintained under the light/dark regime at 25°C. After 2 weeks, adventitious shoots were counted. A shoot was considered adventitious and counted if a stem and leaf developed. Shoots developed along the entire length of the hypocotyl sections. Mean shoot production per hypocotyl explant ranged from 2.4 to 6.1 shoots when seedlings were germinated and grown in 24 h darkness and 2.2 to 10.9 shoots when started in the light/dark regime. Highest shoot counts were attained /from hypocotyl explants when seedlings were germinated and grown under the light/dark regime for 2 weeks and transferred to 2.22, 4.44, or 8.88 μM BA. Shoot development appeared normal at the 2.22 and 4.44 μM level, while at 8.88 μM BA, development was slightly abnormal along with slightly more callus production.