Studies on the cryopreservation of shoot apices from recalcitrant-seeded Trichilia emetica Vahl. and Trichilia dregeana Sond.

Abstract

In contrast to orthodox seeds, recalcitrant seeds are short-lived, shed at relatively high water contents (WCs), and are desiccation sensitive. Presently, the only option for long-term conservation of genetic resources of such plant species is by cryostorage in liquid nitrogen (LN; -196°C) or in the vapour phase over LN (at -150⁰C to -160⁰C). A number of cryopreservation protocols applied for recalcitrant zygotic embryos or embryonic axes of

tropical/sub-tropical species have reported survival as either root or shoot development or callus formation, with no shoot or root production after cryopreservation. This is a consequence of the challenges encountered when optimising the WC for successful cryopreservation across species. Other shortcomings may also be the formation of ice or the sensitivity to desiccation resulting in lethal damage or poor re-growth. However, for successful cryopreservation, a normal plantlet with a shoot and a root needs to be obtained post-cryo. Specimens required for successful cryopreservation must be small; therefore embryonic axes excised from seeds have been often used as the explants of choice. However, in some cases, excised embryonic axes of mature recalcitrant seeds are too large to be cryopreserved, or, even if small, may be adversely affected by excision, dehydration and/or immersion in LN, thus failing to produce plantlets after cryopreservation. As a result, in such cases, there is a need to develop explants alternative to zygotic axes such as buds derived from in vitro shoots, shoot meristems, or shoot apices and somatic embryos. These alternative explants must have a high capacity for plantlet formation before and after cryopreservation. The present study aimed to successfully cryopreserve shoot apices of Trichilia emetica and T. dregeana, tropical recalcitrant-seeded tree species, and monitor the responses or effects of some of the procedural steps involved in cryopreservation on the survival and shoot production from these shoot apices. The main foci of the investigation were to produce vigorous plantlets after

cryopreservation and ultimately develop a protocol for the successful cryopreservation of germplasm of these species. Furthermore, this study reports on a number of factors that may affect survival after cryopreservation, viz. WC of the explants, PVS2 treatment, production of reactive oxygen species (ROS) and levels of endogenous total aqueous antioxidants (TAA) during the various steps of cryopreservation. The effects of the various steps of cryopreservation on the ultra-structure of the shoot apices were also observed. Cathodic protection (by using highly reducing cathodic water; CW) of the explants was attempted to improve vigour and shoot production from the surviving shoot apices after cryopreservation as cathodic water has been reported to ameliorate the excessive burst of ROS, which often accompanies the stresses imposed by the procedural steps of cryopreservation. Experiments were also performed to optimise the medium for vigorous shoot formation from the shoot apices. Shoot apices of T. emetica in this study had an initial WC of ca. 2.2 g g -1 dry weight (DW) upon excision. Although the WC of the shoot apices decreased slightly after cryoprotection with PVS2, it did not result in sufficient dehydration before cooling. Upon retrieval from LN, 68% of the shoot apices survived and 40% of those produced shoots. Treatment of shoot apices with CW did not improve the survival or shoot production from the apices following cryo-retrieval. This could be a direct consequence of increase in WC of the shoot apices following CW treatment. Water content is not the only factor affecting successful cryopreservation; the production of ROS and the level of antioxidants may also have an impact on regrowth after cryogen exposure. Rapid changes in temperature when the samples are cryo-stored and then rewarmed result in an increase in ROS production, which could have affected the shoot production. More importantly the antioxidant activity showed a rapid decrease during recovery,

especially in the CW treated shoot apices, which might have also led to the poor survival and shoot production from the shoot apices. Ultrastructural observations showed the injurious effects of PVS2 treatment typified by derangement of plastids, development of numerous small vesicles along the cell membrane and abnormalities in the structure of the nuclear envelope in the shoot apical cells both before and after cryogen exposure. Following cryo-retrieval, the meristem cells were extensively deteriorated – indicating non-survival, however, some shoot apices had areas of surviving cells which might have led to 40% shoot production after cryopreservation. Based on the studies on optimising medium composition for shoot formation from the apices, woody plant medium (WPM) with 1 mg L-1 BAP + 0.1 mg L-1 IBA was found to be the best medium which gave a higher shoot production of 67 – 70% before cryopreservation compared with only 18 – 20% shoot formation on media used previously. Therefore, this medium was used as the recovery medium. Encapsulation-dehydration of the shoot apices and the use of PVS3 instead of PVS2 for cryoprotection were also employed in an attempt to improve the survival and shoot production after post-cryo, but both methods did not result in any shoot production although 92% and 90% of the shoot apices survived cryogen immersion, respectively. While the shoot apices of T. emetica resulted in 40% shoot production following retrieval from LN and recovery on WPM with 1 mg L-1 BAP + 0.1 mg L-1 IBA, attempts to further improve the shoot production were not successful. The results of this study suggest that the shoot apices used were possibly not sufficiently developed, and with the commensurately high WC, proved to be unsuitable explants for germplasm conservation of T. emetica. The injurious effects of PVS2 treatment both before and after cryogen exposure as observed from the ultra-structural studies provide a clue to the repeated failure to cryopreserve embryonic

axes of many tropical recalcitrant-seeded species after treatment with PVS2. Maintaining mother material in culture for longer durations before explant excision in order to allow better development of the axillary buds and render the cytosol more concentrated, and optimising the exposure duration to loading solution and concentration of sucrose in the loading solution might however, provide sufficient dehydration tolerance to PVS2 leading to successful vitrification up on cooling.