IN VITRO SHOOT MULTIPLICATION OF Feronia limonia (L.) Swingle IN A CO 2 ENRICHMENT AND SUCROSE REDUCTION CULTURE

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1 IN VITRO SHOOT MULTIPLICATION OF Feronia limonia (L.) Swingle IN A CO 2 ENRICHMENT AND SUCROSE REDUCTION CULTURE Enni Suwarsi Rahayu 1) and Noor Aini Habibah 1) 1) Department of Biology, Faculty of Mathematic and Sciences, State University of Semarang, Indonesia *Correspondence enni.bio@gmail.com ABSTRACT This present study was conducted to develop a non-conventional method of Feronia limonia (L.) Swingle shoots multiplication. The research was carried out experimentally using a completely randomized factorial design with two factors, i.e. sucrose concentrations of culture media (0%, 1%, 2%, and 3%); and CO 2 levels of culture vessel (0.0%, ambient concentration, 0.03%, and 0.30%). Explants used in the research were cotyledonary nodes of sterile seedlings. Some variables observed include number of shoots produced in each explant, shoot height, internode length, and the number of composite leaves generated in each shoot after being cultured for three months. Data were analyzed in two-way Analysis of Variance and Duncan s Multiple Range Test. Results showed that sucrose concentrations, CO 2 levels, and their interaction significantly influence the multiplication of F. limonia shoots. Murashige and Skoog medium containing 2% sucrose under 0.03% CO 2 level was the best treatment. It resulted in the highest number of shoots/explant, the tallest shoot, as well as relatively large amount of composite leaf and shoot. CO 2 could replace a part of sucrose in the medium of F. limonia culture that used the cotyledonary node explants under red and blue light during the day and a temperature of 26 ± 2 C. Keywords - shoot multiplication, Feronia limonia (L.) Swingle, sucrose, CO 2 -enrichment Introduction In vitro plant propagation is aimed to produce pathogen-free and high viable plantlets efficiently when transferred to ex vitro environment. The conventional in vitro techniques often face obstacles of a highly contaminated culture and low survived plantlets during acclimatization. This condition is believed to becaused by the sugar usage as a carbon source in the culture medium. This phenomenon results in low photosynthetic activity and reduction of plantlet adaptation in the natural environment. In addition, the presence of sugar in culture media also allows fungal and bacterial contamination. The nutritional type, which chlorophyllous tissue uses endogenous dan exogenous carbohydrates as a carbon source, is called photo-mixotrophy (Kozai and Kubota, 2005). In order to overcome this problem, a different condition of nutritional type is required to produce optimally photosynthesizing plantlets. One way to achieve this condition is by lowering sugar concentration of the culture and replacing the carbon source with a highly concentrated CO 2. The enriching of CO 2 will not only replace the sugar as the carbon source, but also can improve microclimate of the culture media that in turn increases photosynthesis capacity and explant biomass accumulation (Qu et al., 2009). Another advantage of the increasing of CO 2 in the culture is improvement of plantlets produced. The plantlets would be more resistant to microbe contamination and more vigorous with stronger rooting system (Xiao and Kozai, 2006; Xiao et al., 2011). The lowering of sugar and enriching of CO 2 in principle decrease the dependence of culture to exogenous carbohydrates and gradually develop photoautotrophic condition, i.e. the ability of explant to grow without additional exogenous carbohydrates and other organic compound (Kozai and Kubota, 2005). The present study was aimed to find out a non-conventional shoot multiplication 231

2 protocol of F. limonia to produce vigorous in vitro shoots. The effect of CO 2 level of the culture vessel and sucrose concentration of the media at varying concentration and their interaction were studied. The results may attribute to studies of the physiological mechanism of plant in the in vitro culture and development of micropropagation methods of F. limonia or other woody plants. Theoretical Review Photoautotrophic culture has long been expanding in many countries (Kozai and Kubota, 2005), but there is little interest in Indonesia so far. The optimization of photoautotrophic multiplication method is important for propagating rare species, e.g. Feronia limonia (L.) Swingle (Jv. kawista). In Indonesia, F. limonia has a high economic value for food and medicine. At present, the population of this species tends to decline. In Java, most of F. limonia grow in District of Rembang, Central Java. According to Office of Agriculture and Forestry, District of Rembang (2012), plants number in 2009 was 1400 trees, but in 2011, it was reduced to 948 trees. In order to prevent the extinction, this phenomenon should be evaluated. Among some factors of the population reduction, decrease of planting area was the primary factor because of the relatively severe propagation of F. limonia (personal communication with the society in Rembang, 2012). The growth of F. limonia seedlings is very slow; on the other hand, the vegetative propagation using stem cutting or grafting is not effective. For this reason, to increase the availability of the F. limonia seedlings, more efficient propagation should be developed, i.e. through a photoautotrophic tissue culture technique. There are several ways to increase CO 2 level in culture vessel, namely, 1) using large culture vessel with forced ventilation to supply CO 2 (Zobayed et al, 2005), 2) using chemicals to produce CO 2 in the culture vessel, such as NaHCO 3 or Na 2 CO 3 (Vyas and Purohit, 2006), or 3) using gaspermeable membrane or plastic film as the cover of the culture vessel (Saldanha, 2013). The usage of chemicals to produce CO 2 gas is a relatively inexpensive and simple manner. Photoautotrophic culture for F. limonia has been developed by Vyas and Purohit (2006). They examined an effect of CO 2 enrichment on shoot multiplication grown in sucrose media and free-sucrose media. The research result was difficult to apply, especially in a tissue culture laboratory that doesn t have lighting comparable to sunlight perfectly which leads to un-optimal photosynthesis. It means that CO 2 may not able to replace sucrose as a carbon source entirely. Therefore, the treatment of gradually lower sucrose content under 3% needs to be done. In addition, CO 2 enrichment treatment is carried out by Vyas and Purohit (2006) through adding Na 2 CO 3, NaHCO 3, KHCO 3 and K 2 CO 3 in various ratios without acid addition, whereas it has widely been known that NaHCO 3 and Na 2 CO 3 efficiently produce CO 2 at acidic condition. Therefore, the application of these chemical substances was recommended in acidic condition, for example, by adding HCl. Set up of CO 2 concentrations in culture vessel can be based on the following reaction: NaHCO 3 + Na 2 CO 3 + 3HCl 3NaCl + 2H 2 O + 2CO 2. The coefficient ratio of NaHCO 3, Na 2 CO 3, and HCl, as well as CO 2 produced by this equation, was 1:1:3:2. Research Methods This study was carried out in Plant Tissue Culture Laboratory of Biology Department, Semarang State University from March to October Explants used in this study were cotyledonary nodes obtained from seedlings with height of approximately 4 cm derived from in vitro germination of F. limonia seeds. The experiment was conducted using a completely randomized factorial design with 232

3 two factors. There were four concentrations of sucrose (0%, 1%, 2%, and 3%) and four CO 2 levels (0.00 g/m 3, ambient concentration, 0.60 g/m 3, and 6.00 g/m 3 ) investigated. The CO 2 levels of 0.60 and 6.00 g/m 3 were equal to 0.03% and 0.30% respectively (Vyas and Purohit, 2006), while the CO 2 ambient concentration in in vitro vessel usually equal to about 400 ppm (0.40 g/m 3 or 0,02%). Each experimental unit consisted of two cotiledonary nodes. Five replicates were prepared for each of treatment. Various CO2 levels were applied in acrylic boxes; each has a volume of 6,500 cm 3 (25cm x 20cm x13cm) with a lid on the upper side (Fig. 1A). To reach the CO 2 concentration of 0 g/m 3 in the culture vessel, solution of 10% KOH was placed in the acrylic box. For each CO 2 level treatment (0.03% and 0.30%), a petridish containing mixture solutions of NaHCO 3, Na 2 CO 3, and HCl was placed inside the acrylic box with the molarity or concentration ratio based on the ratio of the CO 2 production reaction (Table 1). The solutions were kept in an open petridish inside the acrylic box to provide CO 2 diffusion maximally and changed every 5 days (Vyas and Purohit, 2003). Ambient CO 2 concentration treatment was made by placed the closed culture bottles outside the acrylic boxes in the growth room. The preparation of MS (Murashige and Skoog, 1962) multiplication media was carried out using a standard method, except the addition of sucrose was given in accordance with the treatment. The media MS supplemented with 2 µm BA, 0,5 µm IBA dan 0,3 µm GA3 was adjusted to ph 6 and autoclaved for 20 minutes at 121 C and 15 psi. The cotyledonary nodes as explants were incised from sterile seedlings and planted aseptically on 30 ml media inside 300 cm 3 bottles (Fig. 1B). Sub-cultures were conducted every month on fresh media. Four bottles of each sucrose concentration treatment were placed into each of sterile acrylic boxes with controlled CO 2 level in accordance with the CO 2 level treatment, and then the boxes were covered tightly to avoid contact with the atmosphere. The acrylic boxes were placed randomly according to the experimental design in an incubation room with a temperature of approximately 26 ± 2 C, and illuminated with red and blue tubular lamps (40 W each) placed 40 cm above the culture rack at approximately lux with a 12 hour photoperiod. Various variables observed at month-3 after culturing include the number of shoots resulted in each explant, shoot height, internode length, and the number of composite leaves (CL) resulted in each explant. A shoot was considered be generated when its length at least 1 cm. The CL number was counted based on the number of petiol (usually it had 3 5 leaflets) grown from shoot. The data collected were analyzed using two-way Analysis of Variance and Duncan s Multiple Range Test. Table 1. Ratio of NaHCO 3, Na 2 CO 3, and HCl Used to Produce Various CO 2 Levels in the Acrylic Boxes Based on the Reaction of NaHCO 3 + Na 2 CO 3 + 3HCl 3NaCl + 2H 2 O + 2CO 2 Compounds ratio (M) Compounds ratio (g/l) NaHCO 3 Na 2 CO 3 HCl CO2 NaHCO 3 Na 2 CO 3 HCl CO2 0,007 0,007 0,021 0,014 0,588 0,742 0,756 0,60 0,068 0,068 0,204 0,136 5,712 7,208 7,344 6,00 * Molecular weight of CO 2, NaHCO 3, Na 2 CO 3, and HCl are 44, 84,106, dan 36, respectively 233

4 A B Figure 1. Culture vessels and explants used in the development of photoautotrophic culture. A) Acrylic box for set up treatment of CO 2 level containing four bottles of each sucrose concentration treatment and one petridish containing the mixture of NaHCO 3, Na 2 CO 3, and HCl solution. B) Two cotyledonary nodes cultured on multiplication medium. Results and Discussion In general, emergence of new shoots from cotyledonary node began at the first month and increased over the time until the third month, except on the explants cultured in sucrose-free media under 0% CO 2 which it began at the second month and then died. The number of shoots/explant generated at month-1 until month-3 in sucrose-free media was fewer than those generated in media containing sucrose, and increasing concentration of sucrose increased growth responses (Fig. 2). Sucrose concentrations influenced significantly on shoot multiplication of F. limonia recorded after three months, especially on the number of shoots/explant, number of CL/explant, and shoot height. Explant cultured in the media containing sucrose developed and grew better than those cultured in the free-sucrose media. These results revealed that sucrose was needed in shoot multiplication mechanism of F. limonia. explant in in vitro condition. Sugar is required as carbon and energy source. The sugar kind and concentration must be determined based on type and age of explant; very young organ needs a relatively high sugar level (Saad and Elshahed, 2012). Among the sugars, sucrose is probably widespread used because it is the most common sugar in phloem sap of many plants (Tayz and Zeiger, 2006), highly soluble in water, and does not inhibit the main of biochemical processes inside cells (Saad and Elshahed, 2012), regulates lateral root formation (MacGregor et al., 2008), and triggers in axillary bud development (Mohammed et al. 2013). This result agrees with those achieved on Wrightia tomentos. Sucrose was still needed in the culture media. Although the increase of CO 2 concentration in culture vessels has been proven to improve the shoot development rate, the unavailability of sucrose has caused browning and mortality of W. tomentos shoots after 30 days (Vyas and Purohit, 2003). However, there are some weaknesses in Sucrose is a type of sugar found in usage of sucrose in culture media. Sucrose Murashige and Skoog (1962) culture media is rapidly hydrolyzed, and autoclaving may formulation. Because of the culture also decrease its concentration because it is condition usually unsuitable for broken down into glucose and fructose. photosynthesis, addition of sugar is essential Although the broken down is advantageous for optimal growth and development of because allows generating an available 234

5 carbon source, but subsequently it enables affect ph fluctuation of media and decreases nutrients absorption by explant when it occurs continuously (Saad and Elshahed, 2012). Additionally, Zobayed (2005) stated that ethylene could be produced and accumulated due to the presence of sucrose in the culture media and disturbs stomata development. As a result plantlets grown in a media containing sucrose have lower photosynthetic capacity than that grown in free-sucrose media. According to Badr (2011), the presence of sugar in the media may redirect amino acids synthesis pathways, decreases intermediate compounds of glycolysis and tricarboxylic acid cycle and then disturbs carbohydrate and nitrogen metabolisms. For those reasons, plantlets were grown in media containing sucrose shows metabolic and physiological imbalances. The CO 2 level highly significant affected the number of shoots/explant, number of CL/shoot, shoot height, and internode length. Under different levels of CO 2, explants showed various responses. Best response was observed on 0.03% CO2. Increase in CO 2 level beyond 0.03% showed the decline in shoots/explant and number of CL/shoot. In addition to its function as a source of carbon, CO 2 has a positive role in the development of in vitro leaves. Under high CO 2 environment, leaves develop better than those in the lower CO 2 condition. Yokota et al. (2007) proved that leaves of Aralia eleta and Phellodendron amurens grown in conventional in vitro culture (with low CO 2 concentrations) are thinner and have an undeveloped palisade layer than those greenhouse-grown plants. However, when CO 2 level exceeds the certain point, leaf growth will be reduced. It can be due to an adverse effect of high CO 2 concentration on chlorophyll biosynthesis (Woltering, 1986). There was a significant interaction between sucrose concentration and CO 2 level. Interaction between the two factors had a significant effect on all of the growth parameters measured (Table 2). The number of shoots/explant increased with increasing sucrose concentration in the same CO 2 level. Explants cultured in the media containing 2% sucrose under 0.3% CO 2 produced 5 CO2 0% 10 ambient CO2 C1-S1 5 C2-S month C1-S2 C1-S month C2-S2 C2-S3 10 CO2 0.03% 10 CO2 0.3% month C3-S1 C3-S2 C3-S month C4-S1 C4-S2 C4-S3 Fig. 2. The increase of the number of shoots/explant every month in various CO 2 levels in the culture vessel and sucrose concentrations in the multiplication media. C1, C2, C3 and C4 were CO 2 level at 0%, ambient, 0.03% and 0.3% respectively. S1, S2, S3 and S4 were concentrations of sucrose at 0%, 1%, 2% and 3% respectively 235

6 highest number of shoots (7.5 shoots) and did not significantly differ from those in the media containing 3% sucrose under ambient air. The lowest one was found on free sucrose media under environment without CO 2. In this condition, explant sustained growth for about one month only and subsequently died (Table 2, Fig. 3A). Explant grown in media containing 2% sucrose under 0.03% CO 2 level resulted in an average of 4.6 CL. It was the most number compared to the other treatments. It was interesting to note that there were a similar number of CL/shoots formed in certain sucrose concentration and CO2 level with that formed in lower sucrose Table 2. Treatments interaction concentration and higher CO 2 level. For example, the number of CL/shoot in the media containing 1% sucrose under ambient air (2.8 CL/shoot) similar with those in media without sucrose under CO 2 of 0.03% (2.7 CL/shoot) (Table 2, Fig. 3B, Fig. 3C). Regardless shoot multiplication in the media without sucrose did not well as compared to that occur in the media containing sucrose. It suggested that the F. limonia culture using cotyledonary node still required sucrose as a carbon source. Media containing 2% sucrose under 0.03% of CO 2 could be stated as the best treatment because it produced a biggest number of shoots/explant, tallest shoots as well as a relatively large number of CL (Table 2, Fig. 3D). Effect of interaction between CO 2 levels in the culture vessel and sucrose concentrations in the media on shoot development recorded after 3 months Variables of shoot development number of shoots/explant number of composite leaves (CL)/shoot shoot height (cm) internode length (cm) S1-C1 0.0 k 0.0 k 0.00 e 0.00 c S2-C1 3.1 i 1.9 j 2.76 c 0.97 a S3-C1 3.7 h 3.0 gh 2.42 cd 0.60 a S4-C1 4.1 gh 3.9 b 2.20 d 0.46 b S1-C2 2.1 j 2.1 j 3.18 b 1.07 a S2-C2 4.4 g 2.8 hi 3.02 b 0.79 a S3-C2 6.0 de 3.5 def 2.72 c 0.61 ab S4-C2 7.3 a 3.7 bcd 2.52 cd 0.54 b S1-C3 4.3 g 2.7 i 3.64 a 1.01 a S2-C3 6.3 cd 3.6 cde 3.74 a 0.82 a S3-C3 7.4 a 3.8 bc 3.70 a 0.78 a S4-C3 7.5 a 4.6 a 3.02 b 0.55 b S1-C4 5.2 f 3.1 gh 3.76 a 0.92 a S2-C4 5.7 e 3.3 fg 3.18 b 0.74 a S3-C4 6.5 bc 3.4 ef 3.04 b 0.71 a S4-C4 6.8 b 3.5 bc 2.72 c 0.61 ab F value 7.14** 8.37** 11.69** 2.42* Remarks: C1, C2, C3 and C4 were CO2 level at 0%, ambient air, 0.03% and 0.3% respectively. S1, S2, S3 and S4 were concentrations of sucrose at 0%; 1%, 2% and 3%. The mean followed by different letter in a column showed a significant difference based on DMRT 5%. ** and * represent highly significant and significant, respectively, at 5% level 236

7 In contrast to the two growth parameters before, in general, the shoot height and internode length decreased with increasing sucrose concentration. Shoot produced on media without sucrose under CO % was highest (3.76 cm) and did not differ significantly from those produced on media containing 0 %, 1%l and 2% sucrose under CO2 level of 0.03%. There was no significant difference of node length between shoots in almost of all of the treatments unless the shoots have grown in media without sucrose or 3% sucrose under 0% CO2 (Table 2). It is believed because mechanisms of shoot and leaf development differ from shoot and internode growth. The formation of new shoot and leaf primordium require oriented cell divisions which need some organic compounds and energy (reviewed in Malinowski 2013); while the increasing of shoot height and internode length is conducted by cell enlargement which need more water then organic A C compound (Reddy et al. 2014). In addition, sucrose in culture media was well capable of a performing optimum growth while glucose and maltose performed well especially in an increasing the nodal number per plantlet (Rahman et al., 2010). Based on the results, it could be stated that the explants were able to grow well in the media with lower sucrose under CO2 enrichment. Shoots developed in all levels of CO2 enrichment were higher than those grown at ambient CO2 levels. It could be due to several roles of CO2 in plantlet growth under in vitro condition. Relatively high CO2 concentration may favor some enzymes kinetics, for example, ribulose-1,5bisphosphate carboxylase. As a consequence, activity of carboxylase increases and oxygenase decreases, leading to carbon assimilation enhancement and photorespiration reduction (Reddy et al. 2010). B D Fig. 3. The appearance of F. limonia shoots cultured in the multiplication media. A) in a free sucrose medium under 0% CO2, B) in a medium containing 1% sucrose under ambient CO2, 237

8 C) in a free sucrose medium under 0.03% CO 2, D. in a medium containing 2% sucrose under 0.03% CO 2 Moreover, the CO 2 enrichment increases that of Vyas dan Purohit (2006). The total chlorophyll content, thick and difference might be because of the explant development of leaf tissues, such as xylem, upper epidermis, palisade parenchyma and used. Because more efficient, the explant used in this research was cotyledonary nodes spongy cells (Mohamed and Alsadon, 2010), that have not yet ready to perform enhances photosynthetic rate (Supaibulwattana et al. 2011), reduces stomatal density which lead to a lower relative water loss from leaves and increases photosynthetic pigment, net photosynthesis and growth (Saldanha, 2013). However, CO 2 is not the only determining factor for photosynthesis. The photosynthesis also requires several other factors such as chlorophyll content, light, and temperature. Light is used as an energy source and was harvested by chlorophyll to synthesize glucose from CO 2. The temperature facilitates enzyme activity in order to optimize the photosynthesis process (Taiz and Zeiger, 2006). As a result, the use of CO 2 in culture is only effective when the explants have optimum levels of chlorophyll and grow in a culture room equipped with adequate light and temperature for photosynthesis. This was proven by culture of Asiatic hybrid lilies; CO 2 enrichment accompanied by an increase in photons of light can enhance leaf area, root and bulblet production higher than that induced by CO 2 enrichment only (Mei-Lan et al. 2003). Our results revealed that CO 2 enrichment up to the certain concentration positively impacted on shoot growth and development. This result was consistent with the findings of other previous reports. The addition of CO 2 concentration in the culture vessel increases the growth of Ipoemoa batatas in liquid media without hormone and sucrose (Yulan and Toyoki, 2006), the active compound production of Artemisia annua L. (Supaibulwattana et al., 2011), and many others. photosynthesis and generate a shoot primordium, whereas Vyas dan Purohit (2006) used shoot clump that has been actively formed new shoot. According to Kozai dan Kubota (2005), theoretically, the initiation stage of the culture still need heterotrophic conditions where pathogen free cultures are established by culturing meristematic tissues. Once the chlorophyllous organs able to conduct photosynthesis are developed, the cultures are ready to move on to photoautotrophic micropropagation conditions. Acclimatization step, where the plantlet resulted from in vitro culture grown in nature environment condition, is a critical factor in the success of in vitro propagation of plant. There are accumulating evidence that the CO 2 -enriched cultures may help acclimatization success because of 1) reducing the physiological changes which required for plantlet growth upon transfer to nature environment (Zobayed et al. 2005), 2) a higher density of stomata of in vitro leaves as a major factor related to desiccation control at the beginning of acclimatization (Hazarika 2006), and 3) a higher capability of photosynthesis of leaves similar to the leaves of plants grown in greenhouse (Saldanha, 2013). According to Mills (2009), leaves of plantlet are highly significant affected due to conditions of in vitro culture. Probably the stem is also affected; it has a smaller effect on the plantlet survival at acclimatization phase. For this reason, the F. limonia shoots produced in this research need further examine to check the survivability during the acclimatization process. The number of shoot/explant produced in the present research was lower compared to In the present study, we verified that by employing Na 2 CO 3 and NaHCO 3 in the 238

9 growth vessel could successfully induce photo-mixotrophic culture resulting in healthy plantlets of F. limonia. The using of the chemical compounds in an acidic condition produced saturating CO 2 and probably prevents the CO 2 depletion inside the vessel during the light period. Thus, the photosynthesis optimally occurred resulting in the obtained growth. The most important characteristic of this technique is simple that reduces laboratory cost. Although an enriched CO 2 culture may significantly aid in the growth of plant tissue cultures, a number of cultural factors (i.e. lighting) also contribute towards the growth observed. Conclusion and Recommendations Sucrose concentration, CO 2 level, and the interaction significantly influenced the multiplication of F. limonia shoots. MS media containing sucrose of 2% under CO 2 level of 0.03% was the best treatment. In the F. limonia culture using cotyledonary explant under red and blue light during the day and a temperature of 26 ± 2 C; CO 2 could replace a part of sucrose but not at all. It is necessary to explore the shoots capability to perform root and then grow the plantlet in acclimatization media before the final recommendation on a wide scale. Ackowledgment The authors would like to thank to General Directorate of Higher Education, Ministry of National Education and Culture of Republic Indonesia for financial support through the competitive research grants in References Badr, A In vitro and Ex Vitro Potato Plantlets (Solanum tuberosum) Metabolic Response to Exogenously Supplied Sucrose: A Metabolomic Approach. Dissertation of PhD. Departement De Phytologie Faculté Des Sciences De L'agriculture Et De L'alimentation Université Laval Québec. Hazarika, BN Morphophysiological disorders in in vitro culture of plants. Sci. Hortic. 108, Kozai, T & Kubota, C Concepts, Definitions, Ventilation Methods, Advantages and Disadvantages. In: Kozai, T, Afreen F, & Zobayed SMA (Eds.). Photoautotrophic (sugar-free media) micropro-pagation as a new micropropaga-tion and transplant production system, Netherlands: Springer. Malinowski, R Understanding of Leaf Development - the Science of Complexity. Plants 2: MacGregor, DR, Deak, KI, Ingram, PA & Malamy, JE Root System Architecture in Arabidopsis Grown in Culture is Regulated by Sucrose Uptake in the Aerial Tissues. The Plant Cell 20, Mei-Lan, L, Murthy, HN & Kee-Yoeup, P Photoautotrophic culture conditions and photosynthetic photon flux influence growth of Lilium bulblets in vitro. In vitro Cell Dev. Biology Plant 39, Mills, D Effect of sucrose application, minerals, and irradiance on the in vitro growth of Cistus incanus seedlings and plantlets. - Biol. Plant. 53, Mohamed, ASM, Mousa, MAA, & Bakhashwain, AAS Effects of Growth Regulators and Sucrose on in vitro Nodal Segments and Shoot Tip Culture of Jojoba (Simmondsia chinensis (Link) Genotypes. J. Bioremed. Biodeg. 4 (7), 1-5 Mohamed, MAH & Alsadon, AA Influence of ventilation and sucrose on growth and leaf anatomy of micropropagated potato plantlets. Scientia Horticulturae 123, Murashige, T & Skoog, F A revised medium for rapid growth and 239

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