Plant Biotechnology Laboratory

– The Plant Biotechnology Project of Rizal Technological University was created through the establishment of a plant tissue culture laboratory under the biotechnology priority thrust of the Philippines Agriculture Modernization Program.   [http://plantbiotechrtu.wordpress.com/]

Researches On Orchids at the Plant Biotechnology Laboratory

The Plant Biotechnology Laboratory –R&D Center of Rizal Technological University has produced a number of researches on orchids, and are already published in the Conference Proceedings on the World Conference on Science and Technology. The said researches focuses on micropropagation and fertilizer supplementation of orchid seedlings. The university is focusing on researches in line with conservation of Philippine orchid species. Some of these researches includes the optimization of the culture medium of orchids in vitro, testing the suitability of media formulation like Murashige and Skoog’s medium, Vacin & Went medium, R Medium and Knudson Formula C medium on the growth of individual orchid species; testing the retardation effect of Abscisic acid on orchids in an attempt to induce dormancy and to preserve or prolong storage of orchid seedlings in vitro; testing of anti-contamination agents like Plant Preservative Mixture TM, Ethyl alcohol and Fungicides to control microbial contamination; the identification of growth performances of various orchid species in vitro, and also supplementation of fertilizer to orchid seedlings in order to grow and flower them within a short period of time. With this researches, it aims to establish a strong foundation in the mass-propagation of disease free orchid planting materials for a fast growing local orchid market, and potentially for an export oriented industry. An efficient orchid propagation protocol will take away the burden and stress imposed on our dwindling natural forests, specially against rampant and indiscriminate plant collections. The project will also support and encourage local orchid breeders to produce more unique hybrids both for the local and foreign market, placing the Philippines back in the forefront of the world orchid industry.

I. Anti-Contamination Efficiency Of Ethyl Alcohol And Fungicide In Embryo Culture Of Dendrobium Cv. “Anching Lubag X Allan Umaki (Orchidaceae)

Different levels of ethyl alcohol (0, 2.5, 5, 10, 20, & 40 % / L media) and Dithane TM, a fungicide ( 0, 1/4. 1/2, 1, & 2 tbsp/L media) were used to determine their anti-contamination capacity when added to modified Knudson C culture media. Dendrobium cv. Anching Lubag x Allan Umaki” protocorms were reflasked to each treatment and observed for occurrence of contaminants, and data was converted into percentage. Results showed that the fungicide was effective in preventing contamination at concentration of 2 tbsp./L media to 1/4 tbsp/L media but was observed to be phytotoxic. Ethyl alcohol, on the other hand, was also effective as an anti-contamination agent and is less toxic. However, even if plantlets are not killed in Knudson C, media supplemented with 5% to 0.625% ethyl alcohol, it restricted the growth of Dendrobium plantlets in vitro compared to the control. In view of this study, the two chemicals are not recommended as an anti-contaminant, other chemicals must still be screened for this purpose.

II. Abscisic Acid-Induced Growth Inhibition Of Dendrobium Cv. ‘Lunsom Green’ (Orchidaceae) In Vitro

III. Growth and Flowering Performance of Dendrobium Cv. ” Anching Lubag X Allan Umaki (Orchidaceae) In Foliar Fertilizer

The effect of a complete fertilizer (Gaviota Foliar 60 19-19-19 TM ) was tested on the growth rate of a hybrid orchid, Dendrobium cv. Anching Lubag x Allan Umaki”. Four treatments were performed and these were control, 1.5 tsp./gal, 1.0 tsp./gal. and 0.5 tsp/gal. labeled at Treatment 1, Treatment 2, Treatment 3 and Treatment 4, respectively. Fertilizer application was done twice a week. The highest average growth rate was observed on orchids treated with 1.5 tsp./gal fertilizer followed by 1 tsp./gal., 0.5 tsp/gal. and control, respectively. There was significant difference between the treatments except for Treatment 2 and 3. The greatest number spikes and flowers were also observed between Treatment 1 and Treatment 2. On the other hand, analysis of variance on the number of flowers between all treatments showed significant difference. In view of the results of this study, the application of 1.5 tsp per gal. is recommended.

IV. Effect of Plant Preservative Mixture Tm (Ppm) on Contamination Rate and Growth of Vanda Sanderiana Reichb. F. Seedlings (Orchidaceae) In Vitro

Plant Preservative Mixture (PPM), a proprietary broad-spectrum preservative or biocide as studied to determine its effectiveness and optimum level / concentration in reducing microbial contamination in orchid culture bottles. Different levels of PPM TM (0, 0.125, 0.25, 0.50, 1.0, 2.0 and 4.0 ml/L media) were added to modified Knudson C media. Unsterilized Vanda sanderiana seeds were inoculated to each treatment. Contamination was observed for each treatment and results were converted to percentile. Percent contamination decreased as preservative mixture concentration in basal media increased. Therefore, the preservative mixture appeared to be effective in minimizing microbial contamination in culture bottles.

V. Germination Performance Of Dendrobium “Lunsom Green” (Orchidaceae) In Murashige & Skoogs, Vacin & Went, And Knudson C Media

In vitro culture media, namely Murashige & Skoogs media, Vacin & Went, and Knudson C were tested to determine their effectiveness and efficiency in germinating the seeds of Dendrobium cv. Lunsom Green, Mature seeds were inoculated into the three media and two others as control, one containing water and agar only and the other with agar and activated carbon, and growth performance was measured after 12 weeks. Murashige & Skoogs media provided the highest shoot length and width of protocorms, followed by Vacin & Went andthen, that of Knudson C. However, Murashige and Skoogs media produced semi-vitrified plants. Control containing agar only and that of agar + activated carbon promoted germination but did not supported the further growth of the seeds into protocorms. Therefore, among the three media, Murashige and Skoogs media appeared to be more suitable in germinating Dendrobium cv. Lunsom Green.

VI. Growth Of Dendrobium Ternatense (Orchidaceae) In Three Different Culture Media: Knudson C., Murashige & Skoog And Vacin & Went

Three different culture media: Knudson Formula C (Treatment 1), Murashige & Skoog (Treatment 2), and Vacin & Went (Treatment 3) were utilized and compared to determine appropriate medium to attain optimum growth of slow-growing native orchid species, Dendrobium ternatense. Germinated seeds were reflasked aseptically and growth response was measured (plantlets chosen at random) after seven months. Data obtained showed that treatment 2 gained the highest values and its length and number of leaves and roots and plant height have significant differences. Treatments 1 and 3, on the other hand, have almost the same results. Generally, they have insignificant differences except in the number of roots. Hence, this study suggests the use of Murashige & Skoog culture medium for propagating D. ternatense in vitro.

VII. Growth Response Of Dendrobium Crumenatum (Orchidaceae) To Knudson C Media With Supplements

Growth response of Dendrobium crumenatum on Knudson C media with supplements: Hormex TM, a rooting hormone (1 ml/L media); HB101 TM, an organic rooting hormone (1 ml/L media), and Gaviota 30-10-10 TM, a fertilizer (1/4 tsp/L media) were determined. Protocorms were reflasked to each medium and plantlets, which were randomly selected, were measured after seven months. Parameters measured were plant heights (roots excluded), number and length of shoots and roots, presence of callus and color of plantlets. The differences between treatments were not statistically significant except in the Gaviota set-up where it resulted in low values. This indicates that the addition of Gaviota TM to a standard culture medium provided an exceeded amount of required nut5itional requirements of the plant thereby presenting phytotoxicity. Generally, ,micropropagation of D. crumenatum could make do withouth the above mentioned supplements.

VIII. Suitability Of Growth Media For Embryo Culture Of Grammatophyllum Scriptum (Orchidaceae)

Three kinds of growth media namely: Knudson C, Murashige and Skoogs, and Vacin & Went were compared to determine th suitable media in the embryo culture of a native orchid species, Grammatophyllum scriptum. Seeds were inoculated initially in Kunudson C, Protocorms reflasked, culture bottles were randomely seleted and seedlings (about 2 inches) were measured biweekly for ten weeks. Results showed that there were no significant diffences in the plant leghth, and number and length of leaves of the plantlets grown in three media. Plantlets grown in Knudson C and Vacin and Went have longer roots. Therefore, Knudson C and Vacin & Went culture media are suitable for in vitro culture of Grammatophyllum.

XI. In Vitro Plant Development Of Orchid Species In Different Culture Media At Ptcl, Rtu: Preliminary Observations

Jovita A. Anit, Carnette C. Pulma, Alex B. Quilang, Norberto R. Bautista and Rachel F. Madera Plant Tissue Culture Laboratory Research & Development Center Rizal Technological University Boni Avenue , Mandaluyong City, Philippines
Email: plantbiotech_rtu@yahoo.com

ABSTRACT

Series of observation on the development in vitro of orchids species in different culture media (Knudson C with banana and coconut water, Modified Vacin and Went, Yamada, R, Gaviota with banana and coconut water and Gaviota with coconut water) were made to determine which among these media is the most suitable for each species. Existing embryo-cultured seedlings (Cattleya sp., Coelogyne sp., Cymbidium atropurpureum, Dendrobium cv. Clomen white, D. ternatense, Grammatophyllum scriptum, Phalaenopsis ambilis, Spathoglottis plicata and Vanda lamellata) in the laboratory from different batches were transflasked and distributed aseptically into bottles containing culture media. Qualitative assessment were made on the response of plantlets on each media using the following indicators: survival, shoot proliferation, shoot formation and elongation, root formation and elongation and shoot: root ratio. Development was evaluated by rating the indictor: 0-no response; 1-poor growth; 2-moderate growth; and 4- prolific growth. The different culture media were ranked by getting the average in the result of the evaluation where 1 is the most suitable and 6 is the least suitable. Results showed that KCbc is the best for Cymbidium atropurpureum, D. crumenatum, D. schuetzei, D. secundum, Grammatophyllum scriptum and D. cv. Clomen white. Meanwhile, D. taurinum, Phalaenopsis amabilis, Coelogyne sp. and Vanda lamellata is suitable for R. On the other hand, D. ternatense grows well in G whereas Spathoglottis plicata in Y. Root growth is evident in D. anosmum when placed in MVW and G and Gb complement in the development of Cattleya sp. Qualitative studies will be made to validate such results.

Keywords: embryo culture, in vitro plant development, culture medium, Philippine orchids, orchid hybrid species

X. In Vitro Study On Callus Induction And Plant Regeneration Of Grammatophyllum Scriptum (Orchidaceae)

Rachel F. Madera, Alex B. Quilang, Norberto R. Bautista, Carnette C. Pulma, and Jovita A. Anit. Conservation of Philippines Orchid Species Project Orchid Tissue Culture Laboratory, Research &Development Center Rizal Technological University Boni Avenue , Mandaluyong City, Philippines
Email: plantbiotech_rtu@yahoo.com

ABSTRACT

Somatic embryogenesis and further plant regeneration were observed using young leaves and nodes of in vitro cultured plantlets of Grammatophyllum scriptum, a native Philippine orchid. The explant was cultured in modified Vacin & Went (MVW) medium supplemented with different levels of auxin. Range of 2,4- dicholorophenoxyacetic acid concentration is from 0 to 10 mM. Callus formation was observed to occur in all auxin concentrations except in MVW with no auxin supplementation. Instead, root formation with the presence of root hairs was observed to occur. Callus formed in the different set-ups were all subcultured after three months in MVW without plant growth regulators. Results show that the callus most suitable for plant regeneration was obtained from MVW supplemented with 2,4- dicholorophenoxyacetic acid.

XI. Acclimatization Of Tissue Cultured Banana Cv. “Lakatan” In Different Organic Potting Mix – Alex B. Quilang and Norberto R. Bautista 

ABSTRACTPlant tissue culture is the fastest and efficient way of propagating bananas ( Musa sp .) however acclimatization of its plantlets is one of the most important stages and a critical period in the process. Banana growth and development is mostly toward those sites showing high organic matter content. For this reason, soil and different commercial organic materials were compared in search for a better potting medium for growth and survival of in-vitro cultured “Lakatan” banana. Initially hardened micro plantlets were removed from culture vessels, dipped in fungicide solution, sorted out, and planted in transparent plastic cups with different substrates (soil and commercial organic potting mix) for forty-five days. Results showed that plantlets grown in Treatment 1/Control (Soil/sand), Treatment 3 (Grow Quick TM), Treatment 4 (Growell TM), and Treatment 8 (Vermi TM) have the highest percentage survival (100%, 95%, 90% and 90%) as well as in the means of growth rate (1.92 cm, 2.10 cm, 1.94 cm, and 2.20 cm) and plant height (22.48 cm, 23.60 cm, 25.76 cm, and 25.80 cm). The abovementioned values have insignificant differences but are highly significantly different when compared to Treatment 2 (Enrico TM), Treatment 5 (Nutriplex TM), Treatment 6 (Plantastik TM), and Treatment 7 (Sagana 100 TM) as revealed in the one-way classification of analysis of variance. Treatments that have provided zero survival usually have high water retention. The said organic potting mixes still have an indication of decomposition and fermentation resulting to mortality of planting materials. Hence, application of organic materials and the regulation of the soil cover show the possibility to favor an adequate environment for root development, consequently promoting survival of banana plantlets.

Keywords: acclimatization, tissue culture, banana, “Lakatan”, organic potting mix

XII. An inorganic fertilizer as a culture medium for Cattleya seedling culture – Carnette C. Pulma , Jovita A. Anit and Alexander B. Quilang 

Cattleya presents a broad spectrum of colors and types making it one of the most in demand orchid group for making corsages and bouquets for special occasions. Orchid growers can breed more hybrids. This requires seed germination and seedling culture. This paper presents a method of utilizing Gaviota TM (19-19-19), an inorganic fertilizer as a culture medium for the culture of Cattleya seedlings. Seeds were germinated on the Knudson C medium and the newly germinated protocorms were transflasked to Gaviota fertilizer medium. Varying amounts of fertilizer (g.l -1) were utilized: 0.50, 1.0, 2.0, 3.0, 4.0. Banana (“Saba” cultivar) 150 g.l -1, 30 g.l -1 table sugar, 1 g.l -1 activated charcoal, 5.0 g.l -1 agar and 150 ml.l -1 coconut water were added to the medium. Results showed that the seedlings generally grew well. Tall plantlets were produced on media containing 2.0 and 3.0 g.l -1 Gaviota TM. The number of leaves among treatments is almost similar. Leaf length increased gradually with an increase of fertilizer concentration, but decrease at 4.0 g.l -1. The highest number of roots were observed on 1.0 g.l -1 and was not statistically different from that on 3.0 g.l -1 Gaviota TM. Nevertheless, there was extensive root growth on 3.0 g.l -1. Increases of fresh weight were directly proportional to the increase of fertilizer concentration up to 4.0 g.l -1 when it started to decrease. The plantlets were acclimatized, planted in community pots and established after that. Therefore, Gaviota TM at 2-3 g.l -1 can serve as an inexpensive culture medium for Cattleya seedling growth.Keywords: Cattleya, seed culture, inorganic fertilizer, culture medium, Gaviota TM

XIII. In Vitro Shoot Proliferation Response Of Musa Cv. “Lakatan” To 6-Benzylaminopurine (Bap) And Thidiazuron (Tdz)

Ann Janel A. Vidallon 1 *, Alex B. Quilang 2, Carnette C. Pulma 2, and Jovita A. Anit 2″Lakatan”, a triploid banana is the most highly priced and popular cultivar grown in the Philippines. Tissue culture provides disease-free and true-to-type planting materials where the important requirements are the culture medium and plant growth regulator (cytokinin). 6-benzylaminopurine (BAP) induces more shoot formation than other types of adenine-based cytokinin. Thidiazuron (TDZ) is one of the most active phenylurea with cytokinin-like activities and more active at low concentration than adenine-type cytokinin for woody and herbaceous plants. Hence, effects of Murashige and Skoogs with BAP and TDZ on banana explants are compared. TDZ (1 and 2 ppm) provided 80% positive response compared to 50% in BAP (10 and 20 ppm). Explants in the first (4 weeks) and second (8 weeks) subcultures on BAP had produced more phenolic compound. Its presence is crucial because it hinders the continuous proliferation and growth of the explant. Calli were formed after 2-3 days in TDZ after four days of inoculation while it took 4 days in cultures with BAP. Explants in TDZ generally had exhibited higher multiplication rate than in BAP after the second subculture. The average number and length of shoots of plantlets in TDZ cultures are significantly different from those grown in BAP. TDZ (1 ppm) induced the highest average number of shoots at 6 cm, followed by TDZ (2 ppm) at 5 cm while those on BAP 10 and 20 ppm have only at 3 and 4 cm, respectively. The average length of shoots obtained at TDZ was highest at 1 ppm at an average of 7.5 cm, then 6 cm at 2 ppm. Microplants in 10 and 20 ppm BAP, however, had developed to 2 and 3 cm, respectively. Therefore, TDZ provided better results than BAP. It will be essential for a faster and more productive banana micropropagation.

Keywords: banana, Musa cv “Lakatan”, in vitro, tissue culture, shoot proliferation, callus, cytokinin, 6-benzylaminopurine, BAP, Thidiazuron, TDZ