| Under the new situation of modern grassland and animal husbandry development in our country,alfalfa industry has encountered unprecedented challenges and opportunities. How to break the double "bottleneck" of yield and quality of alfalfa,has been an urgent problem. Crown bud is a major source of alfalfa regeneration, of which the occurrence and development directly determines alfalfa production, persistence, and winter survival. But so far it‘s still a lack of systematic and in-depth study of on crown bud development and dormancy during winter. To find out the winter cold tolerance mechanism and developmental pattern of crown buds, can provide new references for high quality varieties selection, germplasm improvement and high-yield cultivation techniques improvement, which is of great theoretical and practical significance. This study selected WL168(fall dormant varieties) and Gannong No.5(non-dormant varieties), to explore the anniversary rule of growth and development of crown bud, which are based on winter dormancy, preformation, heterogeneity of growth and development and vegetative growth phase changes. The results are follows:1) Crown bud of alfalfa was a complexty of primordium, consisted of growth cone, leaf primordia,young leaves,scale leaves and axillary bud primordia. Descendant buds had occurred from axilla of scale and young leaves before crown buds emergenced from soil. The growth of descendant bud from middle nodes of crown bud was faster than the base and top, and the middle descendant bud quickly differentiated out its own next generation bud before emergence. Therefore, in the same growth cycle, the crown bud born in the base of a shoot was preformed earlier than the shoot began to sprout aboveground. However, crown bud‘s growth and developmental duration varied with their nodes. From the perspective of bud ontogeny, crown bud belonged to axillary bud. Crown buds had to go through 5 stages, brown budâ†' white budâ†'green budâ†'immature shootâ†'mature shoot, and eventually develop into adults. The stage of brown bud and white bud completed underground, green bud emergence and it‘s subsequent development finished aboveground. 11 nodes of the shoot sprouted from crown bud were preformed underground, and when the number of aboveground nodes approximately reached 17, inflorescence primordia and flower primordia occured on the top growth cones.2) Due to differences in occurring time before winter and growing location, crown buds showed heterogeneity during spring regrowth stage. Deep crown buds occured earlier than shallow buds, and then had a high degree of preformation. After deep buds emergence from soil, the development of top cone went into the stage of flowering transition. In contrast, shallow buds occured later, only a part of leaves in vegetative phase was preformed before emergence from soil. Approximately at 6-leaf-stage, the shoots sprouted from shallow buds began to go into the stage of flowering transition. The shallow buds with lower cold resistance were much easier to incur freezing injury during winter.3)The phytomer of alfalfa showed two types of patterns of development: type-I and type-â…¡. Type-I was featured by that compound leaf primordium developed faster than axillary bud primordium, which appeared after the formation of lateral leaflet primordia; whereas for type-â…¡, axillary bud primordium grew faster than compound leaf primordium, initiating after the formation of stipule primordia. Before crown bud emerging from soil, phytomer developed with the type-â… pattern, and instantly after emergence, phytomer‘s development switched to type-â…¡.During development of phytomer, the phyllotaxis of crown buds also showed two different types. Durng the developmental period underground, phyllotaxis was orthodistichous, after buds emergence from soil, the number of aboveground phytomers was up to 11, phyllotaxis turned helical. When divergence angle reached 137°, development of phytomer was accelerated, apical meristem went into the stage of floral organ development. The development of phyllotaxis had a synchronistical relationship with phytomer. We concluded that the shift of phytomer development pattern from type-â… to type-â…¡could be an indicator of transition from vegetative to reproductive growth in alfalfa, and that alfalfa had completed preformations of vegetative organs before crown buds emerging from soil in spring, and then immediately got into the transitional phase of reproduction.4) In the course of crown shoots development, the vegetative growth phase could be divided into 3 periods: early vegetative growth, medium-term vegetative growth, the late vegetative growth(flowering transition). In the stage of early vegetative growth, phytomers were preformed underground, leaf length, petiole length and internode length were all shorter than other stages. In the stage of medium-term vegetative growth, phytomers were preformed just before emergence. Phytomer developed with the type-â… pattern. Phyllotax was orthodistichous; Those phytomers became the most important aboveground vegetative organs after mature. Leaf length, petiole length, internodes length and leaf area were all up to the maximum. During the stage of flowering transition, phytomers were formed aboveground, and developed with the type-â…¡pattern. Phyllotaxis turned helical. Leaf length, petiole length, internodes length and leaf area became all smaller than before.5) By comparing the wintering bud regeneration of two varieties, it‘s found that WL168 could go through physiological dormancy stages during late autumn and early winter, which was divided into 4 stages: induction of dormancy, development of dormancy period, deepened dormancy and released dormancy. Fall dormancy was performance that meristem growth gradually slowed down during the stage of induction of physiological dormancy. Gannong No.5 without physiological dormancy became into the eco-dormancy under the influence of low temperature during late autumn and early winter, elongation of the crown buds restarts in early February the following year. By contrast, WL168 did not show the similar situation until early March.6)A series of active changes in cellar ultrastructure adapted to cold temperature had taken place during overwintering. The concrete show of those changes was: plasmalemma invaginations, the large central vacuole segmentation into multiple small vacuoles, chromatin condensation, plastid becoming crescent or horseshoe-shaped, starch grains depletion or even disappearance,plasmolysis, etc. As a result, the capacity of cold resistance in buds was improved gradually by the adaptive adjustment of cellular ultrastructure. The cold resistance development of white bud during winter went through 4 phases: enhanced phase(late Oct.-mid Dec.), maintenance phase(late Dec.–mid Jan. of next year), decline phase(late Jan.–late Feb.) and termination phase(early Mar.–mid Mar.). By contrast, shallow buds and deep buds of Gannong No.5 show distinct adaptation in the winter. Shallow buds are sensitive to temperature change in the end of October. By the end of November, plastids of the lamellar structure in some shallow buds disappeared, frost damage increase. On January 25 the next year outer membrane mitochondria and plastids appear dissolved. By contrast, deep buds were not sensitive to temperature changes in the end of October. By the end of November, deep buds began to appear cold reactions: a slight plasmolysis, nucleus became small, plastids accumulated starch grains, can survive safely the winter.7) The nuclei and nucleoli in cell of crown bud became smaller when WL168 occured physiological dormancy, the nuclear chromatin condensation, which contained DNA transcription and m RNA synthesis. Meanwhile, plasmodesmata contracts led to the endoplasmic reticulum separated from the channel of plasmodesmata, and then gate of the channel was closed by invaginations of the plasma membrane around. In the end of January, plasmodesmata began to recover. By contrast, Gannong No.5 crown bud cells there had been not such changes. Analysis showed that chromatin condensation and plasmodesmata contraction were cytological mechanism of which caused physiological dormancy. During late autumn and early winter, crown buds of WL168 just went through these changes to improve cold resistance.8) Comparison of changes of anti-oxidation system in crown bud during dormant period of WL168 and Gannon No.5, the results showed that enzyme activity showed up and down as temperatures droped and recoveried during the winter between two varieties. However, before the arrival of low temperature(December 20), enzyme activity of WL168 had reached a very high level after physiological dormancy. WL168 had accessed strong ability to resist to cold in winter. Therefore, the changes of antioxidant system between dormant and non-dormant varieties were different during winter. After cold acclimation, crown buds of dormant varieties due to the mutual induction and regulation between dormancy and antioxidant system, obtained a highly active antioxidant system before the arrival of winter low temperature stress, eventually improved the ability to winter hardness... |
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