REJUVENATION OF WOODY PERENNIALS BY GRAFTING OF ADULT SHOOT APICES ONTO JUVENILE TISSUES IN VITRO AND ITS UNDERLYING MECHANISM
Plant rejuvenation, or phase reversal, can be achieved by repeatedly grafting shoot apices from adult plants onto juvenile seedlings. The procedure results in progressive reappearance of juvenile traits with successive times of grafting. The rejuvenation is attainable in vivo, but accomplishable more quickly in vitro. This investigation is being conducted collaboratively with Dr. Guang-Yuh Jauh, Mei-Chu Chung, Dr. Teh-Yuan Chow, Dr. Yuh-Jang Shieh, Dr. Tsung-Che Tseng and Dr. Grace Shu-Chen Chen in our Institute and Dr. Hao-Jen Huang of National Cheng-Kung University and Dr. T. Murashige of the University of California, Riverside, USA in order to understand the underlying mechanism.
GENERAL OUTLINE
Rejuvenation of trees is evident by restored organogeneses, leaf morphology and shoot vigor, and diminished leaf absicission, chlorosis and tissue and culture medium discoloration in vitro. In our procedure the number of repeated grafts needed to achieve a desired level of rejuvenation varied according to plant genotype. For example, restoration of 100% rooting competence of shoots of Sequoia sempervirens required 4 grafts, whereas that of Citrus reticulata cv. Ponkan mandarin needed 7 repeated grafts. Investigation of the underlying mechanism, using in vitro grown shoots, disclosed identical agarose gel electrophoretic patterns of mitochondrial DNA extracted from juvenile and rejuvenated shoots and distinct from adult shoots. Identity of specific bands from juvenile and rejuvenated was confirmed by double digestions with endonucleases, Southern hybridizations and DNA sequencing. We recently discovered that the differences involved small, circular mtDNA, or plasmids. Mitochondria of juvenile phase shoots contained four plasmids that were absent in those of adult shoots. Furthermore, repeated grafting of adult shoots onto juvenile shoots resulted in the same four plasmids appearing in mitochondria of rejuvenated shoots. No plasmids were 42 43 Annual Report 2005 detected in either undigested chloroplast (cpDNA) or nuclear DNA (nucDNA) preparations. Southern hybridization occurred between mitochondrial plasmid and master-circle mtDNA and among mitochondrial plasmids, but not between mitochondrial plasmids and chloroplast or nuclear DNA. We conclude that the mitochondrial plasmids are vrghlypossible the self-replicating determinants found in the cytoplasm that plays a regulatory role in phase change regulation.
PROGRESS AND ACCOMPLISHMENTS
Preliminary studies on the interrelationship among mitochondrial, chloroplast and nuclear DNA by gel electrophoreses disclosed the presence of small plasmids only in mitochondria, not in chloroplasts or nuclei, more over the gel revealed the similar patterns of small plasmids betweent the juvenile and rejuvenated but different in the juvenile or rejuvenated to the adult in mitochondria. Proteins isolated from various organelles of mitochondria, chlorplasts, or nuclei with 2D PAGE also illustrated the same zymograms between the juvenile and rejuvenated but not the the juvenile or rejuvenated to the adult. Northern analyses indicated the complementary occurrence between mtDNA and mtRNA. The comparison of growth substances especially on ABA and ethylene, and photosysnthetic and respiratory activities all showed the same characteristics between the juvenile and rejuvenated, but different from the adult. Methylation of mtDNA and nuclear DNA specific AFLP also indicated the same methylation pattern between juvenile and rejuvenated shoots while different from the adults. Juvenile or adult specific genes are under identification and sequencing by subtraction hybridization and microarray examinations.
Huang, L. C. 2005. Rejuvenated Sepuoia sempervirens plants from in vitro established transferred to The Experimental Forest, National Taiwan University at Sitou.
Huang, L. C. 2005. Plants established from somatic embryogenesis of Cinnamomum kanehirae in vitro were transferred to The Experimental Forest, National Taiwan University at Sitou.
Huang, L. C. 2005. Plants established from in vitro shoot apical meristem culture and twin scale bulb sections of Lycoris aurea were transferred to the Taiwan Agriculture Research Institute and Private Bai-Yen Agriculture Farm.
Huang, L. C. 2005. Plants established from in vitro propagation of shoot tip culture of Ficus lyrata were transferred to The Experimental Forest, National Taiwan University at Sitou.
Huang, L. C. 2005. Plants established from in vitro shoot apical meristem culture and twin scale bulb sections of Hippeastrum equestre were transferred to The Experimental Forest, National Taiwan University at Sitou.
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