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Malus hupenensis var. `Pingyitiancha' is an important apple stock with many good characteristics, including waterloggig resistance, cold resistance, salt resistance and so on. The three group gene-HVA1 come from barley was transformed into `Pingyitiancha' mediated by Agrobacterium tumefaciens and transformed regeneration plants were obtained in this research. The HAV1 gene cloned from plasmid containing it (offered by Dr. Guo Weidong) by PCR with high fidelity pfu Taq DNA polymerase. It was ligated between BamH 1 and Sac 1 site in PUC118 vector, and identified by electrophoresis after digested with BamH 1 and Sac 1. Through nuclear sequence detecting, it is confirmed that the HAV1 gene cloned in this research is 703bp.This fragment was ligated with 11kb fragment from pB121 plasmid and constructed pBHA vetor. The pBHA vector was introduced in A.tum LBA4404 by triparental mating and the binary vector was obtained. It is cinfirmed that HVA1 gene had been insert in T-DNA by in situ hybirdization. Using `Pingyitiancha' shoot apex, mediated by A. tum. System, the HAV 1 gene was transformed into the plant. Kam resistance regeneration plants were obtained, 6 of them were confirmed as transformation plants by PCR and dot blot.
`Pingyitiancha' has good agricultural traits and is an important rootstock for apples. It was studied for Kam, Cef and Carb concentrations, precultural times, active medium of A.tum, Ca++ concentration, mixed inoculation times of AgNO3, and PVP delay selection times for getting transgenic `Pingyitiancha' stock with more resistance to drought and salt. It was demonstrated that the highest transformation frequency was obtained with 200 mg·L-1 Cef as an A.tum restraining antibiotic, 8 mg·L-1 Kam as a selection antibiotic, GCJ8 active medium, a precultural of 2 days, 5 Ca++ concentrations in preculture and coculture medium compared with standard MS medium, immersed in A. tum as OD600 for 0.5 to 1 min., 4 mg·L-1 AgNO3 and 0.7% PVP as an anti-oxidation compound to reduce hydroxybenzene oxidation and delay selection for 5 days by using `Pingyitiancha' apex shoots as explants. Using mediate of A. tum strain LBA4404 under conditions mentioned above and same explants, the HVA1 gene were transformed into `Pingyitiancha' under the control of CaMV35S promoter and obtained Kam resistance regeneration plants. Six transgenic plants were confirmed by PCR and subsequent dot blot methods.
Cyrtopodium paranaense is a tropical terrestrial orchid, which propagates mainly through sexual seed germination. In this study, we document the asexual morphogenesis of the root tip to protocorm-like body (PLB) conversion in Cyrtopodium paranaense. Protocorm-like bodies sporadically developed from root tips of flask-grown seedlings in the absence of any exogenous plant growth regulators (PGRs). The compact PLBs ultimately gave rise to normal plantlets. Histological observations revealed that the root cap became dissociated from the root apex at an early stage followed by dispersed extension of root vascular strands into nascent PLBs. Protocorm-like bodies also developed from the root central stele tissue. In root tip segment cultures, PLBs were not formed without providing PGRs but were efficiently formed from root tips in Murashige and Skoog (MS) medium supplemented with 10.2 μM indole-3-acetic acid (IAA) and 9.0 μM thidiazuron (TDZ). Both IAA and TDZ promoted the formation of PLBs; however, TDZ did not induce PLB formation in the absence of IAA, indicating a synergistic effect of the two PGRs. Protocorm-like bodies were proliferated and subsequently plants regenerated in PGR-free MS medium. Root tip culture may be used as an alternative method for the propagation of Cyrtopodium paranaense.