EFFECTS OF WATER QUALITY, pH AND STATE OF THE MEDIUM ON GROWTH AND DEVELOPMENT OF COCONUT EMBRYOS IN VITRO By

The effects of the quality qfwater, pH and state qf the medium on growth and development of coconut embryos in vitro were evaluated. Germination of embryos in Y3 liquid medium was not affected by neither wau ,r quality nor pH adjustment. These suggest that tap water could replace glass distilled water and gelling agent could be eliminated in embryo culture qf coconut. However, pH qf the medium has to he adjusted.


INTRODUCTION
One of the types of coconut (Cocos nucifera L.) is the Makapuno coconut which is characterized by a soft enclosperm that fills the nut. Embryos of Makapuno coconuts do not germinate in situ clue to this abnormality of the enclosperm. The team of de Guzman of UPLB clemonstrated the possibility of extracting the normal Makapuno embryo and growing it in vitro to produce true-to-type, high Makapuno yielding coconut palms (de Guzman & del Rosario, 1964;Balaga & de Guzman, 1971a;Rosario and de Guzman, 1976).
The PCA-ARC tissue culture team further improved the technology by looking into the other media formulations to support growth and development of coconut embryos in vitro (Rillo & Paloma, 1990). Using Murashige and Skoog (MS) and Eeuwens (Y3) mineral formulations supplemented. with activated. charcoal, germination was hastened. Altogether the incubation was shortened from 6-8 months to 4-6 months.

REVIEW OF LITERATURE
Water in tissue culture researches should be given great attention since it constitutes 95% of the nutrient medium. Tap water contains high levels of unwanted ions. Therefore, it is passed through a series of purifying filters to free the water of contaminating ions, especially cations (positively charged ions), organic contaminants and even micro-organisms. For research purposes, water that has been glass distilled is recommended, while for research the photoplasts, cells and meristems, water which has been double distilled (Pierik, 1987).
The pH of the nutrient medium should also be given. an important consideration for a successful in vitro growth performance of any crop. It is predicted that a pH in the range of 5.0-6.5 is suitable for growth. pH lower than 4.5 or higher than 7.0 generally stops growth and development of cultures in vitro (Pierik, 1987). Butenko (1968) reported that if the pH is too low the agar becomes too, sloppy; phosphate and iron salts may precipitate; Vitamin 131 (thiamine and aneurine) and pantothenic acid become less stable; and uptake of ammonium ions is retarted.
The state of the medium is also important. Based on preliminary studies on the growth and development of zygotic coconut embryos in vitro, there was longer and greatbr primary root formation in Y3 solid than in Y3 liquid, MS solid or MS liquid media. Secondary roots were already evident in seedlings cultured in Y3 solid medium. The average number of scale leaf formation, however, was the same in Y3 solid, Y3 liquid and MS solid. After another four weeks in culture, it was observed that there were more formation of expanded leaf and greater ramification of secondary roots in Y3 solid medium than in the other media (PCA-Annual Report, 1990). However, since sampling was not enough to really come up with conclusive statements, a follow-up study on the effect of the water source, pH, and state of the medium was conducted.

OBJECTIVES
Generally, this study aims to determine if mass propagation cost of Makapuno using embryo culture could be reduced.
Specifically, it aims to investigate the effects of water quality, pH and state of the medium on growth and development of coconut embryos in vitro.

MATERIALS AND METHODS
Embryos were excised from 10-11 month-old nuts collected from Laguna Tall palms. They were cultured following the protocol established by Rillo and Paloma (1992). The treatments were assigned completely at random in a three-factor experiment using single-factor experimental design. The following factors were considered: Water quality (W) Wl glass distilled water W2 tap water pH (P) PI adjusted to 5.8 P2 not adjusted State of the medium (S) Sl solid S2 liquid Treatments were replicated 4 times with 20 samples per replicate.
Germination of embryo, shoot and root lengths were recorded after one month in culture. Plant height and further root elongation were regularly measured, 2, 3, and 4 months after initial culture.
Data were analyzed using ANOVA. Means were compared using the Duncan's Multiple Range Test (DMKr).

DISCUSSION OF RESULTS
Statistical analysis showed that quality of water and pH of the media affected root length but not percent germination and shoot length of l0 to 11 month-old Laguna embryos ( Fig. l a & b) after one month in culture. Longer roots and shoot lengths were observed in embryos germinated in Y3 liquid media which used glass distilled water or pH adjusted to 5.8 (Table 1). However, their interaction effect was not significant.
After a 2-month incubation, the increase in plant height was significantly affected by state and pH of the medium but not by the quality of water. Although not significantly different, the increment in plant height was higher in tap water. Higher increment in plant height was observed in Y3 solid than in liquid medium. Likewise, adjustment to ph to 5.8 resulted to taller seedlings. The increment in root length of the seedlings was not significantly affected by any of the variables tested ( Table 2).
Incubation of the seedlings in their respective medium for three to four months showed that only pH significantly affected plant height (Table 2, Fig. 2). The effect of pH in terms of increment in root length was observed only after 3 months. This effect, however, became negligible after the fourth month in culture. No interaction effect among the three factors was detected.  Chemical analysis of the tap water collected from Albay Research Centre showed that it contained 4.76 ppm potassium, 26.0 ppm calcium, 10.8 ppm magnesium, 14.4 ppm sodium, 2.0 ppm chloride, 2.4 ppm sulfur and nil amount of nitrogen, phosphorus and boron. From the results, it is safe to assume that the ions present in the tap water did not significantly affect growth and development of coconut embryos. The pH of the Y3 media which used glass distilled water and tap water ranged from 4.8-5.0 and 6.2-6.4, respectively (Table 3). Skirvin et al (1986) reported that pH changes after autoclaving. If the starting pH is in the range of 5.0-7.0, it generally lowers by 0.3-0.5 unit. The pH range of 5.0-6.5 is predicted suitable for growth with an optimum at about 6.0 (Pierik, 1987). Hence, assuming a reduction of 0.3-0.5 units in pH due to autoclaving, the pH of the autoclaved culture media which used tap water with or without pH adjustment was within the predicted suitable range for growth (Table 3). The pH after autoclaving of culture media which used distilled water could be below the pH range for growth if pH was not adjusted prior to autoclaving. 6.2-6.4 -5.7-5.9 a -Ph range assuming a reduction in pH by 0.5 units.
The cost of producing an in vitro-cultured Makapuno in a solid medium with glass distilled water and pH adjustment, as previously applied for coconut embryo culture, was found expensive (P150.35). Comparing the cost of the previous method (W1P1S1) with the other treatments suitable for embryo culture, the amount saved was P3.61, P15.23, and as much as P18.84 with W1P1S2, W2P lSl and W2PlS2, respectively (

CONCLUSION
Percent germination of 10 to 11 month-old Laguna embryos cultured in Y3 liquid medium was, not affected by either water quality or pH adjustment. However, further culture of the coconut embryos showed that pH adjustment is a critical factor. The quality of water and state of the medium are not very important factors. The culture medium for coconut embryos could, therefore, use tap water, without agar but with pH adjustment. The elimination of agar and the use of ordinary tap water will greatly reduce the production cost of in-vitro cultured Makapuno coconuts.

RECOMMENDATIONSTOLICY IMPLICATIONS
The results of this study influenced partly the policy of Philippine Coconut Authority to reduce the selling price of in vitro grown Makapuno seedlings from P 500.00 to P300.00 per piece, thereby, allowing more small coconut fariners to afford this high value coconut type. Sociologically and economically, the availment of this type of coconut will benefit the farmers in the form of being able to sell high value coconuts helping them in the short to medium term.