Effects of plant growth regulators on the rapid proliferation of shoots and root induction in Spath

Effects of plant growth regulators on the rapid proliferation of shoots and root induction in Spathiphyllum floribundum cv. Maya White LIU Jian-fu1, 2, LI Xue3, WANG Ming-yuan1, FAN Yan-ping21.Department of Biological Engineering & Technology, Huaqiao University;2.College of Horticulture, South China Agricultural University, Guangzhou;3.Sunshine Horticulture co, LtdE-mail: jianfu@AbstractAn efficient plant regeneration protocol was developed for rapidly propagating Syngonium podophyllum cv. Maya White, via shoot organogenesis. Shoot multiplication was induced on (MS) medium supplemented with 3-Indoleacetic acid (IAA), 6-benzylaminopurine (BA), kinetin and α-naphthaleneacetic acid (NAA). Rooting was induced on full- or half-strength MS medium supplemented with NAA and indolebutyric acid (IBA) and BA. The maximum mean number of shoots (3.2) was obtained from a single explant by the combined effect of 5.4 μm NAA and 13.3 μM BA. The longest roots and a minimum number of roots were produced in the medium without plant growth regulators. The shorter roots and the largest number of roots were observed in the medium supplemented with 2.7 μM NAA and 4.4 μM BA.Keywords: Spathiphyllum floribundum cv. Maya White; In vitro culture; Rapid Propagation1. IntroductionSyngonium is a genus of about 36 species of flowering plants in the family Araceae, native to tropical rain forests in Central and South America. They are woody vines growing to heights of 10–20 m or more in trees. They have leaves that change shape according to the plant's stage of growth, and adult leaf forms are often much more lobed than the juvenile forms usually seen on small house plants. Syngonium species are often grown as house plants, usually only in the juvenile foliage stages.Consequently, there is a growing commercial demand for Syngonium podophyllum, which has caused such serious problems as unrestricted exploitation and nonselective cultivation, leading to the dwindling of wild sources and species degeneration. Additionally, conventional propagation of Syngonium podophyllum through seeds is low due to poor germination. In vitro micropropagation can provide a more efficient approach for the rapid mass propagation of selected elite Syngonium podophyllum varieties. However, to date, there have been only a few reports on the micropropagation of Syngonium podophyllum[1-4].The present study was carried out to find an efficient micropropagation protocol for highquality commercial Syngonium podophyllum products, and conserving the existing germplasm.2. Materials and Methods2.1. Plant material and preparation of explantsTwigs were collected from 2 year old trees of Syngonium podophyllum cv. Maya White, grown in the germplasm repository at Sunshine Horticulture co, Ltd, Quanzhou City, Fujian Province, China.Eight-month.-old seedlings grown in the glasshouse at 27/15±2℃ day/night temperature were used as a source of explants. Actively growing shoot tips (1.0-1.5 cm long) were marinated in distilled water for 30 min, and washed three times with sterile distilled water, then sterilised in 75% ethanol for 0.5 min, and 9% Domestos bleach (containing, 4.25% w/v available chlorine) for 25 min with gentle stirring. Shoot tips were rinsed three times in sterile distilled water. The young leaves and basal portions of shoots injured by sterilant were removed and 0.8–1.0 cm long shoot-tip explants were cultured. 2.2. Culture mediumThe basic nutrient medium consisted of MS[5] medium with 3% sucrose and vitamins, adjusted to pH 5.7 with 0.1 N NaOH and solidified with 0.6% agar. Each 60 ml jar contained 15 ml medium and was sterilized by autoclaving for 20 min at 121℃, 105 kPa. All cultures were incubated at 26±1 ℃ and were exposed to a 12 h photoperiod under 45 µmol·m-2·s-1 quantum flux density provided by cool daylight fluorescent tubes with 80 % ± 5 % relative humidity maintained in the culture room.2.3. Shoot proliferationIn the first experiment, freshly prepared explants were cultured on medium containing 6-benzylaminopurine (BA) (4.4, 8.9, 13.3 μM) and 3-Indoleacetic acid (IAA) (0.6, 2.9, 5.7μM). In the second experiment, similar explants were cultured on BA (4.4, 8.9, 13.3 μM) and α-Naphthaleneacetic acid (NAA) (1.6, 2.7, 5.4μM). In the third experiment, similar explants were cultured on BA (4.4, 8.9 μM) and NAA (1.6, 2.7, 5.4 μM) and kinetin (KT) (6.9 and 9.3 μM) to evaluate the effect on shoot proliferation. All the treatments were added 0.6 % (w/v) agar and 3 % (w/v) sucrose. The pH was kept at 5.6-5.8 prior to autoclaving. Observations were recorded on morphogenetic characters such as days taken for bud break, number of axillary shoots per explant, number of leaf per explant and length of axillary shoots after 30 days of culture of the explants. Thirty explants were used for each experimental unit. Data on survival, number and length of shoots were recorded at 3 wk intervals in shoot-proliferation experiments.2.4. RootingActively growing shoots with four or five nodes from the second subculture produced in medium with 13.3μM BA + 5.7 μM IAA were used for rooting. Initial experiments showed no spontaneous rooting when shoot tips were cultured on MS medium supplemented with no growth regulators. Thus rooting medium consisted of MS macro- and micronutrients at full and half strength with indolebutyric acid (IBA) at concentrations of 1.5, 2.5, 5.0, 7.5, 10.0 μM, or NAA at 0.5, 1.6 and 2.7 μM, supplemented with 3 % sucrose and agar (0.55% or 0.6%). Cultures were maintained in a growth room under the conditions described above. Observations were recorded on per cent rooting, number of roots, length of roots, diameter of roots and weight of roots. Thirty explants were used for each experimental unit. Rooting experiments were evaluated at 6 wk, and data were recorded on the number of rooted shoots, and the number and length of roots.2.5. Hardening and establishment of plantlets in the greenhousePlantlets were transferred into small plastic pots filled with peat, perlite and sand (1:1:1; v/v), and were kept inside the seed propagator of a culture room at 25–30℃, 12 h photoperiod. Each plantlet was fed twice a week with 10 ml 10.7 mM NAA，and then transferred to a greenhouse at 30–40℃, RH 70%, 12 h photoperiod after 4 wk.3. Results and discussion3.1. Shoot multiplicationThe effect of combination with BA and IAA on shoot induction auxiliary shoot multiplication was shown in Table 1. The concentration of growth regulators in the medium influenced markedly on shoot regeneration. The 100 percent Shoot formation, the maximum leaves/explant (2.5) and highest shoot length (3.37 cm) were found in BA 13.3μM and IAA 5.7μM, significantly different to the medium of no plant growth regulator. However, the results of the present study indicated that NAA is unsuitable for in vitro propagation of Syngonium podophyllum, as most treatment showed on the medium containing NAA showed poor proliferation and shoot length (Table 2). Addition of the same concentrations of NAA and BA to the medium did not improve the rate of shoot proliferation in Syngonium podophyllum (Table 2). This result is in agreement with that of Omura and Hidaka (1992), who found that the addition of NAA is not as critical as that of BA for shoot-tip culture of Satsuma mandarin. The reason may be due to the ability of tissues to synthesize the required amount of auxin endogenously (Smith and Murashige, 1970).Table 1.Effect of BA and IAA on shoot induction and development of Syngonium podophyllum cv. Maya White after 4 weeks ofcultureBA(μM)IAA(μM)No. ofshootsperexplantShootlength(cm)No. ofleavesShootformation(%)4.4 0.6 2.3 3.03 2.0 83.34.4 2.9 2.2 3.13 2.1 94.44.45.7 2.0 2.90 0.9 70.98.9 0.6 2.2 3.00 1.8 82.48.9 2.9 2.4 3.33 1.4 99.18.9 5.7 2.1 3.07 1.7 85.5 13.3 0.6 2.4 2.83 2.2 93.2 13.3 2.9 2.1 3.23 1.8 86.7 13.3 5.7 2.5 3.37 2.5 100Axillary shoots produced under all treatments, and shoot length did not increase with further subculture on the same medium. Individual shoots were difficult to separate due to lack of shoot elongation. Concentrations of kinetin significantly affected the length of adventitious shoots (Table 2). The length of shoots increased with increasing concentrations of kinetin. Application of kinetin in the medium produced significantly fewer shoots per explant, and these shoots were very long, thin, and etiolated.The data in Table 3 revealed the effect of different concentration of sugars on shoot regeneration in Syngonium podophyllum cv. Maya White. Shoot proliferation and shoot length at 2.5% concentration was found to be maximum i.e., 2.3 and 3.13 cm, respectively (Table: 3). In case of sucrose, 2.5% concentration shows the maximum shoot length. shoot growth were promoted by the lowest concentration (2.5%). Present findings show variations against earlier observations recorded by tissue culturists [6].Table 2.Effect of BA, NAA and kinetin on shoot induction and development from of Syngoniumpodophyllum cv. Maya White after 4 weeks of cultureBA (μM) NAA (μM) KT (μM) No. of shoots per explantShoot length(cm)No. of leavesShoot formation(%)4.4 1.6 0 1.6 2.67 1.3 93.1 4.4 2.7 0 1.5 2.57 1.1 96.4 4.45.4 0 2.0 2.77 1.7 90.7 8.9 1.6 0 2.2 2.77 1.2 88.4 8.9 2.7 0 2.1 3.07 0.5 71.9 8.9 5.4 0 2.5 3.27 0.6 75.8 13.3 1.6 0 3.0 2.63 0.7 82.3 13.3 2.7 0 2.6 3.07 0.8 86.5 13.3 5.4 0 3.2 2.60 0.7 80.0 4.4 1.6 6.9 1.6 2.94 0.7 82.2 4.4 2.7 6.9 1.5 2.93 0.9 95.9 4.4 5.4 6.9 1.6 3.00 0.9 91.3 8.9 1.6 9.3 2.4 3.21 1.0 98.8 8.9 2.7 9.3 1.4 3.18 1.2 96.6 8.95.49.31.32.331.194.2Table 3.Effect of sucrose concentration onshoot induction and development from of Syngonium podophyllum cv. Maya White after 4 weeks of culture (MS + 6BA13.3μM +IAA0.6μM )Sucrose concentration(%)shoots per explant Shoot length (cm) No. of leaves Shoot formation(%) 2.5 2.6 3.13 2.5 96.1 3.0 2.3 3.05 1.9 87.9 4.02.22.791.882.5Fig.1.Effects of PGRs on shoot induction anddevelopment of Syngonium podophyllum cv.Maya White in vitro shoots High adventitious shoot proliferation efficiency was induced by high concentrations of plant growth regulators (PGRs). However, the establishment of a regeneration system should take into consideration the relationship between proliferation increment and plant quality to ensure the most economical production of high quality seedlings. The enrichment culture medium MS + 13.3 μM BA + 5.7 μM IAA was utilized in all further studies.3.2. In vitro rooting of isolated shootsAfter the 4 week subculture, rooting of shoots was carried out on a full- or half-strength MS medium supplemented with different concentration of NAA or agar (Table 4). In most treatment, root initiation appeared when the shoots were rooting easily succeeded when the shoots were transferred to the rooting medium after 10 days. The rooting was affected by the strength of basal medium and the NAA concentration. However, rooting percentage was lower on a full strength MS than half-strength MS medium at the same concentration of plant growth regulators. The half-strength MS medium supplemented with 2.7μM NAA and 0.6% agar was the best for rooting induction. All plantlets intended to produce roots after 10 days (Table 4). Rooting percentage was lower in full strength MS than in half-strength medium, which has been reported on some other ornamental plant.The optimal medium for root differentiation was determined using various combinations of auxins, the combination of BA + NAA, and BA + IBA at different concentrations. Roots were induced after four weeks of culture with the best root formation obtained in MS medium containing NAA, and significant difference in root formation were observed under PGRs treatments (Table 5). The maximum number of roots was observed in MS supplemented with 4.4 μM BA + 2.7 μM NAA, which each shoot regenerating a mean of 5.4 roots, significantly higher number compared to the other media used (Table 5). The smallest number of roots was observed in the hormone-free medium (each shoot regenerated 1.3 roots). The result showed that the highest root length was recorded from 4.4μM BA + 1.5μM IBA.3.3. Plant establishmentThe highest transplantation rate (89.5%) wasobtained when plantlets were transplanted to planting beds containing a mixture of peat, perlite and sand (1:1:1; v/v) in a greenhouse. After 2 months, the surviving plantlets were transplanted in plastic pots. Compared to mother plants, the in vitro raised plants were not showed any phenotypic variation using our safe and effective propagation technique after planting.Table 4.Effects of various MS salt concentration and NAA on the rooting of Syngonium podophyllum cv. Maya White in vitro shootsMedium NAA(μM)Agar(%)Shoot height(cm)Root-ing(%)No. of rootsper shootRoot length(cm)MS 0.5 0.55 4.1 70 5.6 5.2MS 1.6 0.55 3.9 82 5.1 3.7MS 2.7 0.55 3.2 64 4.3 2.3 1/2MS 0.5 0.6 3.4 73 4.5 5.0 1/2MS 1.6 0.6 4.1 85 5.4 4.9 1/2MS 2.7 0.6 4.5 96 4.9 5.4Table 5.Effects of the combination with various concentrations of BA, IBA and NAA on the root induction of Syngonium podophyllum cv. Maya White in vitro shootsBA(μM) NAA(μM) IBA(μM)Shootheight(cm)Root-ing(%)No. of roots pershootRoot length(cm)4.4 0.5 0 27 63 3.6 2.2 4.4 1.6 0 35 85 4.1 3.7 4.4 2.7 0 30 935.4 3.0 4.4 0 1.5 31 25 3.5 4.3 4.4 0 2.5 35 29 1.3 1.9 4.4 0 5.0 29 31 0.9 1.4 4.4 0 7.5 25 73 3.6 2.2 4.4 0 10.0 32 82 4.1 3.7 0 0 0 32 27 1.3 2.3Fig.2.Effects of PGRs on the rooting of Syngonium podophyllum cv. Maya White invitro shootsFig.3.Plantlets of Syngonium podophyllum cv.Maya White 4. ConclusionThe results obtained were valuable because of the possibility of establishing shoot cultures and further scale-up. A complete and rapid clonal propagation system was established for Syngonium podophyllum cv. Maya White using auxiliary bud. The regeneration frequency (3.20-fold in shoot induction) on MS medium supplemented with 5.4 μm NAA and 13.3 μM BA. The entire procedure could be completed without callus formation, which could increase possible genetic variability caused by callus formation. The high rates of multiplication and rooting and successful transfer to a poly bag make this production system useful for exsitu conservation and large-scale multiplication of Syngonium podophyllum cv. Maya White.References[1].Liu Yi-qing, Liu Chang-chun, Xu An-hui. 2007.Effects of sucrose concentrations on the growth and physiology of test-tube plantlets of Syngonium podophyllum cv. Golden Allusion. Journal of Southwest University (natural science edition) , 29(6):149-152[2].Wang Shou-ming, Piao Xuan-chun, Lian Mei-lan, etal. 2004. Effects of BA and IBA on the propagation and rooting of Spathiphyllum floribundum CV．Cupidtest-tube seedling. Journal of Agricultural Science Y anbian University, 26(2):128-131.[3].Zhu Gen-fa. 2003. Tissue Culture and Propagation ofSpathiphyllum. Chinese Agricultural Science Bulletin, 19(3):75-77.[4].Teixeira J.A., Silva D.A., Giang D.D.T., et al. 2006.Photoautotrophic micropropagation of Spathiphyllum.Photosynthetica, 44(1):53-61. 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