This review paper highlights the influence of bud break and apical meristematic tissue competence in inducing embryogenic tissue during cloning mature trees of conifers. Bud burst depends on dormancy release by chilling or heat due to increase in the day temperature, and accumulation of thermal time above a species-specific threshold. The buds collected immediately after the bud burst were found very responsive for the in vitro cloningof P. kesiya, P. roxburghii and P. wallichiana, P. patula, and P. sylvestris, This might be duethe activation of the apical meristematic cells showing active growth of shoots in most of theconifers. The active dividing and totipotent cells might be positioned only at the cambiallayer of the apical meristematic tissue in conifers, so that their growth and division leads to acontinuous flow of progeny cells. These cambial layer cells under stress conditions undergodifferentiation and leading to the embryogenic pathway in conifers

B. Malabadi, Ravindra

Kumar, S. Vijaya

Nataraja, K.

S. Mulgund, Gangadhar

T. Meti, Neelambika

English

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 Research in Plant Biology, 2(2): 43-47, 2012                                                    ISSN : 2231-5101  www.resplantbiol.com      Short communication Influence of bud break and apical meristematic tissue competence during cloning mature trees of conifers  Ravindra B. Malabadi*, Gangadhar S. Mulgund1, Neelambika T. Meti2, K. Nataraja1, S. Vijaya Kumar3  1Department of Botany, Karnatak University, Pavate nagar, Dharwad-580003, Karnataka, India 2Department of Agricultural Biotechnology, Bharati Vidyapeeth University, Pune-Satara Road, Katraj, Pune - 411046, Maharashtra, India 3Department of Biotechnology, Madanapalle Institute of Technology and Science, Madanpalle- 517325, Chitoor, Andhra Pradesh, India *Corresponding author e-mail: mlbd712@rediffmail.com        This review paper highlights the influence of bud break and apical meristematic tissue competence in inducing embryogenic tissue during cloning mature trees of conifers.   Bud burst depends on dormancy release by chilling or heat due to increase in the day temperature, and accumulation of thermal time above a species-specific threshold. The buds collected immediately after the bud burst were found very responsive for the in vitro cloning of P. kesiya, P. roxburghii and P. wallichiana, P. patula, and P. sylvestris, This might be due the activation of the apical meristematic cells showing active growth of shoots in most of the conifers. The active dividing and totipotent cells might be positioned only at the cambial layer of the apical meristematic tissue in conifers, so that their growth and division leads to a continuous flow of progeny cells. These cambial layer cells under stress conditions undergo differentiation and leading to the embryogenic pathway in conifers.  Key words: Apical meristem, bud burst, cloning, conifers, mature trees, somatic embryogenesis   Overview   Pine bud break (burst) timing is very important factor, which influences the in vitro cloning mature trees of conifers. The timing of bud break of pines is also varied from one location to another location area within the country or from one country to another country within the same continent or different geographical location throughout the world. Survival of pine species depends on synchronization of their annual growth-dormancy cycle, bud break or burst with local climate. The developmental or growth cycle from bud set to bud burst consists of several sub processes-dormany induction, dormancy release and forcing-each with different responses to climatic conditions. Buds undergoing dormancy in winter, cessation of growth in the fall and initiation of growth of buds during early summer are key processes in this life cycle of conifers (Choudhury, 1962; Konar, 1958, 1963; Dormling et al. 1968).  Therefore, temperature and change in the climate of a particular location influences bud burst timing to a greater extent. In species with a free growth pattern, growth cessation  Malabadi et al. / Research in Plant Biology, 2(2): 43-47, 2012  44  and cold acclimation are induced by short day and low temperatures in fall.  After growth cessation, bud dormancy develops gradually and a state of true dormancy may result after 8-10 weeks. True dormancy can be defined as the inability of a bud to burst at normal growth temperatures in long (Weiser, 1970; Hanninen et al. 2007). After dormancy induction, bud burst depends on dormancy release by chilling or heat due to increase in the day temperature, and accumulation of thermal time above a species-specific threshold. In some of the plant species e.g. black alder (Alnus glutinosa L) and birch (Betula spp), increased temperature in autumn enhances the chilling requirement and delays bud burst, lowering the risk of premature bud burst in later winter and early spring, a risk that may increase in the event of climate warming (Heide, 2003). The timing of cold hardening in plants, onset of dormancy and bud burst are genetically determined that are adapted to local conditions. In case of conifers or trees with a wide range of distribution, the timing of bud set and the development of frost resistance can vary significantly between different provenances or ecotypes of the same species (Choudhury, 1962; Konar, 1958, 1963; Dormling et al. 1968; Heide, 2003; Weiser, 1970; Hanninen et al. 2007; Repo et al. 2000).    In India, there are 4 clear seasonal changes that is rainy season (1st of June to 30th of August), fall (September to November), winter (November to February), summer (March-May) respectively. During winter season (November- February), the shoot buds of P. kesiya, P. roxburghii and P. wallichiana were found dormant and growth of the shoot buds was inhibited by the low temperature in both North and Southern parts of India. The bud burst might be during the early days of summer, showing the first sign of active growth in March or some times in April (depending upon the climate change for the particular year). The buds collected immediately after the bud burst were found very responsive for the in vitro cloning of P. kesiya, P. roxburghii and P. wallichiana, P. sylvistris, and Lodgepole pine (Aronen et al. 2007, 2008; Aronen, 2009; Malabadi et al. 2004; Malabadi and Nataraja, 2006; Malabadi and Nataraja, 2007; Park et al. 2009). This might be due the activation of the apical meristematic cells showing active growth of shoots in most of the conifers. In P. roxburghii the shoot buds collected after the bud break during April have a positive response in terms of inducing embryogenic mass of tissue. In P. kesiya, apical shoot buds induced somatic embryo-genesis during the period of April-July. This is due to a change of climate during the particular year during the collection of buds for in vitro cloning studies of P. kesiya. In P. wallichiana, the shoot buds were collected during the month of May has yielded an embryogenic tissue (Malabadi et al. 2004; Malabadi and Nataraja, 2006; Malabadi and Nataraja, 2007). Apical meristematic cells may retain a high enough degree of juvenility to make them more amenable to  in vitro cloning of mature conifers.  The active growth of apical shoots of P. kesiya, P. roxburghii and P. wallichiana immediately after the bud burst is only a very short window (for the collection) for in vitro cloning studies. This short period of availability of plant material limits the scale of investigation. The buds collected after bud burst are very active in growth and also successfully induced somatic embryogenesis in P. kesiya, P. roxburghii and P. wallichiana (Malabadi et al. 2004; Malabadi and Nataraja, 2006; Malabadi and Nataraja, 2007).  On the other hand shoot buds collected during entire year in our in vitro cloning of mature pines has resulted in the induction of non-embryogenic tissue might be due to the absence of  active internal regulatory signals for somatic embryogenesis. In P. patula, the shoot bud material was available during end of March to middle of April and resulted in  Malabadi et al. / Research in Plant Biology, 2(2): 43-47, 2012  45  the induction of embryogenic tissue mass (Malabadi and van Staden, 2003; 2005a, 2005b, 2005c; Malabadi and van Staden, 2006). In rest of the collections of the whole year, the apical shoot buds of P. patula from South Africa resulted in inducing a non-embryogenic tissue (Malabadi and van Staden, 2003; 2005a, 2005b, 2005c; Malabadi and van Staden, 2006).   This is also holds good for P. sylvestris from Finland where the bud material is available only during the month of April-May (summer started in Finland). The shoot buds collected before April or after May has yielded the non-embryogenic tissue due to a short window of availability of plant material (Aronen et al. 2007, 2008).     Therefore, the optimum time for the collection of plant material particularly apical shoot buds for in vitro cloning of mature pines is immediately after the bud burst or break, which is always influenced by local climate change in temperature due to the onset of summer season in many countries of the different geographical locations throughout the world. This time frame of availability of bud material is very short, and needs to be identified through the study of growth cycle for a particular pine species before starting the in vitro cloning experiments. Although latitudinal patterns of variation have been found in angiosperm trees such as oak (Quercus spp.) and birch, the effect of latitude is often confounded with the climatic gradient. It was also estimated that the predicted rise in temperature in this and next century could advance the time of bud burst by 2 to 3 weeks in Norway spruce growing at 64o N, which could increase the risk of injury by spring frosts and indicates the need to identify late-flushing genotypes for planting in areas where spring frosts frequently occur (Hanninen et al. 2007).    During our studies on in vitro cloning of differnt pines (P. kesiya, P. roxburghii, P. wallichiana, P. patula, and P. sylvestris), the storage of plant material at 2 to 4°C for 3 days have not affected the induction of embryogenic tissue. On the other hand, storage of bud material more than 3 days has drastically affected the induction of embryogenic tissue, and resulted in the browning of tTCL and discarded. This might be due to a over stress conditions of the bud material during the long-term storage of the bud material.   Therefore, for a better and optimum results, the storage of shoot buds either in cold or room temperature should be avoided. Induction of somatic embryogenesis using vegetative shoot apices of mature pines was very successful without storage of bud material in the most of pines e.g. P. kesiya, P. roxburghii, P. wallichiana,  P. patula.   In all our experiments, shoot buds were processed for in vitro cloning on the very same collection day, and this has influenced the success rate of in vitro cloning to a greater extent than storing the bud material in terms of higher percentage of responsive explants (tTCLs) in inducing embryogenic tissue. Another reason is that during storage, most of the buds were wounded; wounding itself induces the stress and resulted in the browning during initiation of embryogenic tissue, and ultimately resulted in the death of the explants. Therefore, storage of buds has negative effect on the tissue initiation and ultimately resulted in the non embryogenic tissue. Therefore, we conclude that storage of shoot bud material at 2 to 4oC for more than 3 days has resulted in the browning of explants leading to the induction of non embryogenic tissue from mature pines. Hence storage must be avoided and the buds should be processed immediately.                In vitro tissue culture conditions expose the explants to significant stresses, as they are removed from their original tissue environment and placed on synthetic media containing non-physiological concentrations  Malabadi et al. / Research in Plant Biology, 2(2): 43-47, 2012  46  of growth regulators, salts and organic components. Wounding itself is a significant signal in the induction of dedifferentiation. Stresses not only promote dedifferentiation, but also can be used to induce somatic embryo formation. Wounding, high salt concentration, heavy metal ions or osmotic stress positively influenced somatic embryo induction in diverse plant species. These procedures were accompanied by increased expression of diverse stress related genes; evoking the hypothesis that somatic embryogenesis is an adaptation process of in vitro cultured plant cells             Plants continuously maintain pools of totipotent cells in their apical meristems from which root and shoot systems are produced. Most plant organs are formed during the postembryonic stages from the meristems. The shoot meristem is the source of all above-ground post-embryonic organs in higher plants. It carries out organ formation by balancing the maintenance and proliferation of undifferentiated totipotent cells, and the direction of these cells towards differentiation. The shoot apical meristems are organized pools of undifferentiated or embryonic cells maintained by a dynamic balance between cell division and differentiation. In case of pines, the transverse thin layer showed outermost epidermis layer, then internal layer of cortex region, followed by thin cambial region and central pith or medullar region respectively. The actively dividing  totipotent cells are positioned only at the cambial layer of the apical meristematic tissue in conifers, so that their growth and division leads to a continuous flow of progeny cells. These cambial layer cells under stress conditions undergo differentiation and leading to the embryogenic pathway in conifers. On the other hand the rest of the layers (epidermis, cortex region and central pith or medulla) have induced non-embryogenic tissue in conifers. References Aronen TS (2009) Initiation of somatic embryogenesis from mature scots pines-microscopical examination. IUFRO Tree Biotechnology, Conference, June 28th- July 2nd 2009, Whistler, BC, Canada, P-107,  p 56 (abstract). Aronen TS, Pehkonen T, Malabadi RB, Ryynanen L. 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Hanninen H, Slaney M, Linder S (2007)  Dormancy release of Norway spruce under climatic warming: testing ecophysiological models of bud burst with a whole-tree chamber experiment. Tree Physiology  27:291-300. Heide OM (2003) High autumn temperature delays spring bud burst in boreal trees, counterbalancing the effect of climatic warming. Tree Physiology 23:931-936.  Malabadi et al. / Research in Plant Biology, 2(2): 43-47, 2012  47  Konar RN (1958) A quantitative survey of some nitrogenous substances and fats in the developing embryos and gamatophytes of Pinus roxburghii Sar. Phytomorphology 8: 174-176 Konar RN (1963) Anatomical studies on Indian pines with special reference to Pinus roxburghii Sarg. Phytomorphology  13:388-402. Malabadi RB, Choudhury H, Tandon P (2004) Initiation, maintenance and maturation of somatic embryos from thin apical dome sections in Pinus kesiya (Royle ex. Gord) promoted by partial desiccation and Gellan gum. Scientia Horticulturae 102: 449-459. Malabadi RB, Nataraja K (2006) Cryopreservation and plant regeneration via somatic embryogenesis using shoot apical domes of mature Pinus roxburghii Sarg. trees. In Vitro Cellular and Developmental Biology – Plant 42: 152-159 Malabadi RB, Nataraja K (2007) Smoke-saturated water influences somatic embryogenesis using vegetative shoot apices of mature trees of Pinus wallichiana A. B. Jacks. Journal of Plant Sciences 2: 45-53 Malabadi RB, van Staden J  (2005a) Somatic embryogenesis from vegetative shoot apices of mature trees of Pinus patula. Tree Physiology 25:11-16 Malabadi RB, van Staden J (2005b) Role of antioxidants and amino acids on somatic embryogenesis of Pinus patula. In Vitro Cellular and Developmental Biology – Plant 41: 181-186. Malabadi RB, van Staden J (2005c) Storability and germination of sodium alginate encapsulated somatic embryos derived from the vegetative shoot apices of mature Pinus patula trees. Plant Cell, Tissue and Organ Culture 82: 259-265. 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Influence of bud break and apical meristematic tissue competence during cloning mature trees of conifers

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Influence of bud break and apical meristematic tissue competence during cloning mature trees of conifers

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