PROPAGATION OF PECAN (CARYA ILLINOENSIS) USING IN VITRO TECHNIQUES

Abstract

During the present work, an in vitro approach was followed for the propagation of Pecan [Carya illinoensis (Wangenheim) C. Koch]. Effect of different media (DKW, MS or WPM) supplemented with various levels of BAP were tested for in vitro germination response. It was observed that the best medium for in vitro germination was an MS formulation supplemented with 4.0 μM BAP. During the experiments, different morphological features of in vitro-grown seedlings of Pecan were also observed. Formation of multiple shoots was also observed from intact nodal regions of developing seedlings. Multiple shoots developed from in vitro germinated seeds were shifted to various rooting media. After acquiring a sufficient length (3 - 4 cm), the developed multiple shoots were transferred to the rooting media, i.e., DKW or MS supplemented with different combinations of growth regulator (IAA, IBA or NAA). MS medium supplemented with 4.0 μM IBA + 4.0 μM NAA proved to be best medium for root induction. On the other hand, in vitro-germinated seedlings after acquiring a sufficient length (4 - 5 cm), were transferred successfully to perlite or vermiculite to enhance rooting. More than 85 % of in vitro-grown Pecan plants were acclimatized successfully to the soil under glasshouse conditions and kept for more than 30 days. These plants were then transferred to field conditions at Botanical Garden, Punjab University, Lahore. For the clonal propagation of Pecan, forcing shoot tips and/ or epicormic buds from the large stem segments taken from more juvenile portions of older trees. Softwood shoots were forced form shoot tips of Pecan during the dormant season. Forcing solution (8-HQC) containing sucrose (2 %), TDZ (2.0 μM) in combination with IBA (2.0 μM) and BAP (2.0 μM) was quite effective for the highest (89.45 %) sprouting of buds under glasshouse conditions. Softwood shoots were also forced through epicormic or latent buds form the large branch segments on different media under various environmental conditions. The present investigation demonstrated that glasshouse conditions favored the maximum (2.92) production of softwood shoots as compared to other lab or wire house (natural) conditions. Media also had a significant effect on softwood shoot production as sterilized sand produced the highest (2.92) mean number of softwood shoots during the winter season. Rooting experiments with these softwood shoots however were not successful as contamination was the major limitation. During the present investigation, a suitable explant for callus induction and its subsequent maintenance was established for Pecan (Carya illinoensis Wan.). Bark segments and immature cotyledons of Pecan were used as an explant source. Effect of different media (DKW, MS or WPM) supplemented with various levels of 2, 4-D, NAA and TDZ were tested for callogenesis from bark and cotyledonary explants. Mature bark explants cultured on DKW medium containing a combination of 2, 4-D and TDZ resulted in 93.70 % callus induction and proved to be the best medium for callus induction and its maintenance. DKW medium supplemented with 13.57 μM TDZ resulted in 93.33 % callus induction from immature fruit explants. Morphologically different calluses were also observed at various levels of 2, 4-D, NAA and TDZ. Tissue browning was a major problem associated with callus induction using bark and fruit explants. These brown callus cultures, however, formed root primordia during this study after an incubation period of 110 days or so. Calluses were also transferred for plant regeneration, however, there were no plantlet regeneration possible during the present investigation. In the present study, adventitious multiple shoots were initiated from immature cotyledonary explants of Pecan (Carya illinoensis). The embryo axes were excised carefully and small cotyledonary pieces from immature fruits were cultured on different media (DKW, WPM or MS) supplemented with various levels of benzyl aminopurine (BAP) or Thidiazuron (TDZ). The shoots were initiated after 8 days from the cultures on DKW or MS media supplemented with BAP (0.5, 1.0, 4.0, 8.0 or 15 μM). Media supplemented with TDZ had shown no response. Maximum shoot development and proliferation was observed after 16 days of culture on MS medium supplemented with 15 μM BAP. The developed shoots were then transferred to basal MS medium for further development and shoot proliferation for 20 days. The proliferated shoots (2.0 - 4.5 cm long) were transferred (without any pre-treatment) to fresh MS basal medium for root initiation. Although rooting could not be achieved during this research work, some progress has been made in this regard. However, adventitious regeneration indicates a strong possibility to regenerate whole plants from various tissues of Pecan.