The Development of Plant Regeneration System from Callus of Pineapple (Ananas comosus L.)

Malaysia’s production of canned pineapples has been decreasing since 1992. Two important factors that have been a hindrance to the progress of this industry are competition from other producers and the increasing demand for fresh pineapples. Current varieties need to undergo qualitative improvements. Genetic modification, breeding and selection are some crop improvement techniques that are not successful at the moment in developing varieties that can replace current world varieties. Somaclonal variation is another technique for obtaining desirable variants, which have been achieved in crops such as sugarcane, wheat and sorghum. Highly stable variants that can be transmitted to progenies, and a more controlled change of their characteristics than those of induced mutations were achieved. However, this technique requires a plant regeneration system from callus cells. These cells have a tendency to mutate, and more cells are mutated under prolonged culture and rapid proliferation, and so generate more variants for selection. Therefore, the objectives of this project are to induce calli, proliferate old calli and regenerate shoot from calli. For calli induction, meristemic globular bodies (MGB) of Moris and Josapine, were cultured in various levels of auxin naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic (2,4- D). The highest percentage of MGB forming calli was observed in treatment NAA 14 and 10 mg/L for Moris and Josapine respectively, at the end of 16 weeks. For calli proliferation, 18 month-old calli were cultured in various levels of NAA, 2,4-D, bnaphtoxyacetic acid (BNOA) and p-chlorophenoxyacetic acid (4-CPA) auxins for 12 weeks, and then in various levels of casein hydrolysate (CH) and coconut water (CW) in the presence of NAA 6 mg/L for 12 weeks. Among the various levels of the four auxins, NAA 6 mg/L proliferated healthy and high mean calli fresh weight. However, NAA 6mg/l supplemented with CW and CH also gave healthy and generally higher mean calli fresh weight than NAA 6mg/L alone. NAA 6 mg/L alone was considered the most economical treatment for calli proliferation, while NAA 6mg/L supplemented with 15%v/v CW and 300mg/L CH gave significantly highest mean calli fresh weight and was considered the best treatment for rapid calli proliferation. For shoot regeneration, 18 month-old calli were cultured in various levels of NAA, 2,4-D, BNOA and 4-CPA auxins for 12 weeks, and then in combinations of various levels of auxins (BNOA and 2,4-D) and cytokinins (Benzylaminopurine [BAP], Kinetin and Adenine) for 12 weeks. Among the various levels of the four auxins, 2,4-D at 1mg/L regenerated high number of shoots, and was considered the best treatment for high shoot regeneration from calli that were considered as high competency calli. However, regeneration response from these calli were gradually decreasing, such that treatment BNOA 6mg/L combined with BAP 1mg/L (with subculture) (that statistically gave highest number of regenerated shoots) and an extended culture period of 12 weeks (without subculture), generated mean number of shoots was considered as not satisfactory. Subsequently, the 18 month old-calli (now 27 months) were cultured in various combination levels of CH and CW for 12 weeks, but these failed to show any regenerated shoots from calli that were now considered low competency calli. However, calli obtained from treatment NAA 6 mg/L + 300mg/l CH + 15%v/v CW (rapid calli proliferation treatment, and now 27 months old), preserved calli competency and regenerated highest mean number of shoots in treatment 10%v/v CW and 200mg/l CH, and was considered the best treatment for regeneration of shoots from low competency calli

RAPD analysis of colchicine induced variation of the Dendrobium Serdang beauty

Variation was detected in Dendrobium Serdang Beauty V (DSB V) plantlets regenerated from protocorm like bodies (plbs) induced by various concentration levels of colchicines in the Murashige and Skoog media (MS) supplemented with 1.5 mg/L IBA. RAPD analysis detected 6 - 26% variation in the regenerants from the mother plant. The highest variation was obtained in regenerates treated with 25 mg/L colchicine, which also exhibited reduced regeneration rates from plbs and mean plantlet fresh weight. RAPD analysis also showed high polymorphism between the mutated regenerant DSB V, and 13 species of the Dendrobiumgenera, and 13 orchids across generas. However, despite the 26% colchicine induced variation in the regenerants, all RAPD analysis revealed that DSB V was closely related to the mother plant. Thus, the RAPD technique is favourable for variation detection as it was sensitive enough to detect variations at species level and among somaclonal variants in this study

Establishment of a plant regeneration system from callus of Dendrobium cv. Serdang Beauty

An in vitro propagation protocol was established for the Dendrobium Serdang Beauty orchid. The propagation protocol utilized calli tissues that were successfully initiated from protocorm-like bodies (PLBs) explants, while the leaf and root tip explants died. The percentage of protocorm-like bodies explants responding to calli formation was 100% in all tested levels of IAA, IBA and NAA auxin treatments. The highest amount of calli (49.59 gram) proliferated on MS medium containing 1.5 mg/L IBA. These calli successfully regenerated on media supplemented with either KIN or BAP cytokinins and combined treatments of KIN and IAA (4 mg/L) or NAA (1.5 mg/L). However, media supplemented with only 1 mg/L KIN was sufficient to produce significantly high percentage of plantlet formation (80%), high number of planlets per explant (4-5 plantlets) and high mean fresh weight per plantlet (11.128 g). These plantlets were acclimatized on all tested media and obtained satisfactory rate of plantlet survival (80-100%), mean number of leaves per plant (4-6 leaves), and mean leaf length (4 - 5 cm). Among these media, charcoal was considered the most economical and available material in the local market. During the development of this protocol, substantial necrosis of calli were observed when cultures were treated with 2,4-D and BAP. It was proposed that the presence of ethylene within the cultures, which is known to be emitted by plant growth regulators into the micro-climate of in vitro culture vessels, is the determining factor of a suitable plant growth regulator for the survival and growth of the Dendrobium Serdang Beauty calli cultures in our study

A modified way of producing humic acid from composted pineapple leaves

Purification of humic acid (HA) is time-consuming (takes between 2 to 7 days). A study was conducted to investigate whether HA produced from composted pineapple leaves could be purified within a day through washing with distilled water. Standard procedures were used to produce 0.1, M KOH and pineapple leaves compost. The KOH was used to extract HA in the compost using standard methods with some modifications. The HA was purified by suspending it in 100 ml distilled water, equilibrated for 1 hour, centrifuged for 15 minutes, supernatant decanted, filtered through glass wool and the liquor analyzed for K, Ca, Mg, Na, Zn, Mn, and Cu using an atomic absorption spectrophotometer (AAS). This procedure was repeated four times after which the washed HA was oven dried at 30°C to a constant weight. Washing HA for four consecutive times within a day was able to reduce the ash content of the HA to 0.1%, a value less than the generally accepted value of less than 1%. This observation was attributed to the remarkable decrease in K, Ca, Mg, Na, Zn, Mn, and Cu with washing. This finding can help in facilitating the production of K-rich humate (organically based fertilizer) from composted pineapple residues in a relatively short time since the HA can be purified within a day for its reconstitution to produce K-humate (38% K) instead of the conventional method that takes between 2 to 7 days. © 2004 by The Haworth Press, Inc. All rights reserved