Factors influencing regeneration and ß-glucuronidase expression from apple (Malus × domestica Borkh.) stem internodes were studied as part of a program to develop transgenic `Royal Gala' apple with improved disease resistance. The early stages of the transformation process were monitored by counting the number of ß-glucuronidase (GUS) expressing zones immediately after co-cultivation of explants with Agrobacterium tumefaciens supervirulent strain EHA105 (p35SGUS_INT) and by counting the number of GUS-expressing calli developing on explants 2 weeks after co-cultivation. Etiolated shoots were produced from in vitro shoots cultured for 2 weeks in the light followed by 2 weeks in the dark and were compared with shoots cultured for 4 weeks in the light (green shoots). First internodes from etiolated shoots produced three, 10 and 100 times the number of shoots regenerated from second, third, and fourth internodal explants, respectively, and produced seven times the number of shoots compared with similar explants from green shoots. 100% of first internodes from etiolated shoots exhibited GUS-expressing zones and yielded twice as many GUS-expressing zones when compared with leaf explants from green shoots, which exhibited GUS-expressing zones in only 60% of the explants. An average of nine GUS-expressing calli per explant were produced on first internodes from etiolated shoots 2 weeks after co-cultivation.
Qingzhong Liu, Sarbagh Salih and Freddi Hammerschlag
Kathleen Heuss, Qingzhong Liu, Rosemarie Hammond and Freddi Hammerschlag
As part of our program to develop transgenic peach cultivars with improved disease resistance, we showed that grafting of in vitro cultured `Suncrest' peach [Prunus persica (L.) Batsch] tips `onto decapitated stems of Prunus necrotic ringspot virus (PNRSV) infected `Suncrest' shoot cultures, resulted in consistent transfer of virus across grafts as demonstrated by RNA hybridization analysis, suggesting that such a system could be useful for measuring resistance to PNRSV in peach shoot cultures. We have extended these studies to include grafts of `Springcrest' and `Nemaguard' test tips onto `Suncrest' stocks. RNA hybridization analysis showed that PNRSV persists in shoot cultures for 18 months after initiation from PNRSV-infected `Suncrest' trees and after 16 weeks of treatment of 4°C in the dark, suggesting that a supply of infected shoot cultures could be maintained for repeated use. Graft success rates for grafts of `Springcrest' onto `Suncrest' and `Nemaguard' onto `Suncrest', equaled or exceeded success rates for `Suncrest' onto `Suncrest'. Virus was transmitted from infected stocks into `Suncrest', `Springcrest', and `Nemaguard' test tips by 2 weeks in most successful micrografts. There was no significant difference in the virus concentrations among the three scions at 2, 4, and 6 weeks after grafting, suggesting that there is equal efficacy of virus transfer through grafts from `Suncrest' to the three cultivars, and that no differences in resistance to PNRSV exist among these cultivars.
Xiaojuan Zong, Jiawei Wang, Li Xu, Hairong Wei, Xin Chen, Dongzi Zhu, Yue Tan and Qingzhong Liu
Improving the poor resistance to environmental stress and the weak development of roots system in the cherry (Prunus) rootstock ‘Gisela 6’ (Prunus cerasus × Prunus canescens) is of great importance for sustainable sweet cherry (Prunus avium) production. Although a stable genetic transformation system has been developed for ‘Gisela 6’ rootstock, there is little information on the identification of genes involved in stress resistance. Using the cherry rootstock cultivar Gisela 6, we identified a total of 12 novel mitogen-activated protein kinase (MAPK) genes, designated PcMPKs. Phylogenetic analysis revealed that the PcMPKs could be divided into four groups, designated A, B, C, and D. In addition, an intron–exon structure analysis for the PcMPKs was conducted to help further understand the structure–function relationships within the cherry family. The expression profiles of PcMPKs in response to abiotic and biotic stresses were characterized using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Five PcMPKs (i.e., PcMPK4-1, PcMPK4-2, PcMPK3, PcMPK6, and PcMPK18) exhibited differential expression, and suggested their potential roles in plant responding to various stresses. This study provides the basis for further analysis on the physiological functions of PcMPKs in environmental tolerance in cherry rootstocks.