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High Temperature Stress Responses and Establishment of Genetic Transformation System of Salvia Splendens

Author: LiuHui
Tutor: ZhangGuoPing
School: Zhejiang University
Course: Crop Science
Keywords: Salvia splendens Ker-Gawl High temperature stress Heat tolerance Two-dimensional electrophoresis Mass spectrometry Plant growth regulator Regeneration Genetic transformation
CLC: S681.4
Type: PhD thesis
Year: 2012
Downloads: 7
Quote: 0
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Abstract


Salvia splendens Ker-Gawl. most commonly used to add a splash of brilliant color in a warm season, frequently encounters heat stress in the South China, and its growth and flowering are greatly affected by high temperature. In the present study, high temperature stress responses were investigated in Salvia splendens to determine the morphological and physiological mechanisms associated with the heat tolerance. Comparative proteomic analysis of Salvia splendens varieties, Vista and Kings under40℃stress for4d were conducted to gain a comprehensive understanding for heat tolerance molecular mechanisms. The effect of various combinations of plant growth regulators (PGRs) and explant types on the callus induction and plant regeneration of Salvia splendens was investigated, and in consequence, genetic transformation system using Agrobacterium-mediated and pollen-tube pathway was studied to provide a theoretical and technical basis for development of heat-resistant Salvia splendens varieties. The main resuts are as follows:1. There was a significantly different in growth response to natural heat stress among of Salvia splendens varieties. Huo Wa and Pround Red had the longer florescence. Plant highth. rown diameter, inflorescence length and number of the examined varieties were significantly affected by high temperature stress. All varieties showed slower growth and a slightly wilting of leaves when the plants were exposed to high temperature. However, the current heat stress did not affect the flowering of Salvia splendens. On the whole, Pround Red, Queen, Huo Wa and Hong Hai showed higher visual values and heat tolerance.2. The variety Shenzhouhong had longer florescence, and its rown diameter, inflorescence duration length were less affected by heat stress than Emperor under naturally high temperature. In a growth incubator with40℃. there was no significant difference in SPAD values (relative content of chlorophyll) between two varieties, while heat stress resulted in significant reduction of Fv/Fm (maximum quantum yield of Photosystem Ⅱ photochemistry) and SOD (superoxide dismutase) activity, and significant increase of CAT (catalase) and POD (peroxidase) activities, as well as MDA (malondialdehyde) and proline contents. Moreover, the affected extent differed greatly between two varieties. It may be suggested that Shenzhouhong had much greater ability than Emperor in the capacity of light protection and active oxygen scavenging system in response to high temperature stress. 3. Heat stress induced reversible inactivation of PS Ⅱ reaction centers and production of protective enzymes and antioxidants, which alleviated oxidative damage of Vista. More than1213protein spots were reproducibly detected on each2-D gel, wherein23and28proteins were differentially up-or down-regulated by heat stress, respectively. There were8pair of spots from the identified proteins similar to both varieties. Most proteins were involved in photosynthesis, metabolism, protein processing and stress response, especially in photosynthesis. Among them, most proteins belong to heat shock protein families, such as the small Hsp (sHsp) family, the Hsp70(DnaK) family and the Hsp90family. These heat shock proteins were responsible for protein folding, refolding, assembly, degradation, translocation under stress conditions, indicating that they could play a crucial role in protecting Salvia splendens against heat stress by re-establishing normal protein conformation and thus cellular homeostasis under heat stress.4. The medium containing BAP (6-benzylaminopurin), NAA (naphthalene acetic acid) and2,4-D (2,4-Dichlorophenoxy acetic acid), could induce callus from stem and leaf explants, whereas the medium containing only BAP and NAA failed to induce organogenic callus from mature seeds. For the two cultivars examined. Queen and vista, the highest rate of callus induction from leaf and stem explants occurred in the medium containing1.0mg·L-12,4-D and1.5mg·L-1BAP, whereas the highest rate of callus induction from mature seeds occurred in the medium containing1.0mg·L-BAP and2.0to2.5mg·L-12,4-D. Although shoots were sporadically initiated at very low frequencies from the calli induced from stem segments with nodes, the calli induced from stem or leaf segments did not form shoots. Many regenerated plantlets were obtained from the cotyledon-derived calli, induced from mature seeds, and the medium containing2.0mg·L-1TDZ (1-phenyl-3-(1,2,3-thia-diazol-5-YL) urea) and1.0mg·L-1KT (kinetin), showed the highest efficiency in shoot regeneration of the two Salvia splendens cultivars.5. A Cyclophilin2gene (Cyp2) was transferred into Salvia splendens by using agrobacterium-mediated method, and it was primarily proved by PCR analysis that the foreign gene was integrated into the genome of transformated Salvia splendens. and thus a few of positively transgenic plants were obtained successfully. In the genetic transformation system, the rate of resistant callus formation could be significantly enhanced under the following conditions:less than30days for callus subculture, OD600=0.2-0.4in the concentration of agrobacterium and10minutes in infection. However, the seed setting rate of Salvia splendens decreased significantly in the transferred plants by pollen tube pathway. Although a few seeds were harvested, no positive transgenic plants were found by PCR analysis. The results was likely to be associated with the soaked time of inflorescences after pollination. Further research is required to set up a more efficient genetic transformation system for Salvia splendens.

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CLC: > Agricultural Sciences > Gardening > Ornamental Horticulture ( flowers and ornamental trees) > Annual and biennial flower class > A bunch of red
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