Indian Journal of Experimental Biology Vol. 39, December 200 I, pp. 1280-1287

Morphogenetic responses of six Philodendron cultivars in vitro

S Sreekumar, S Mukunthakumar & S Seeni*

Plant Biotechnology Division, Tropical Botanic Garden and Research lnstitute,Palode, Thiruvananthapuram 695 562, India.

Received 12 March 2001; revised 27 August 2001

In vitro morphogen ic response of nodal explants from six cultivars of Philodendron viz, blue mi sti c, painted lady, pink prince, pluto, royal queen and green emperor was studied. Frequency and number of shoot formation depend en the cultivars and concentrat ion of BAP. High frequency and number of shoot formation were obtained w hen the nodal explants were cultured in Nitsch med ium suppl emented with BAP (6.8- 11.8 J1M), sucrose (2%) and agar (0.45%), initially in the dark for 8- 10 weeks followed by 16 hr photoperiod. Regenerated shoots were rooted on medium without growth regulators. After two weeks of hardening, rooted and rootle s shoots were establi shed in the soil with more than 90 and 65% survival rates re­spectively, while the unhardened pl antlets showed a much lower percentage (20%) establishment. A standard protocol for the rapid multiplication of Philodendron is presented.

Philodendrons are of great interest due to their indoor and outdoor decorative value. Among the foli age plants they constitute a major share in both domestic and ex port market and their cost varies from few rupees to few thousands per plant in loca l nurseries. Conventional propagation of Philode11.dron by stem cuttings and seeds is slow and inconsistent with the demand . Micropropagation is the best option to solve thi s problem by providing consistent supply of quality plant materials at a des irable pace. Although many private firms are actively involved in the producti on and supply of Philodendrons , the protocols are known only to them and as such are not published. Ti ssue culture studies on some spec ies of Philodendron reported earli ert -4 essenti ally deal with bud proliferation and rooting in vitro bu t a rapid propagation protocol that could be used by others fall short of required demand. It is particularly so since morphogenic response in ti ssue culture is also t.lepcndent on the genotype or the mother pl ant and variati ons in response may occur, even between cultivars7

·8

, that abound among Philodendrons. In the present study we have in vesti gated the in vitro regeneration response of six culti vars of Philodendrons to different cytokinin treatments and have also developed mi cropropaga ti on protocols for all of them.

Materials and Methods To study the genotypic effect, S IX cultivars of

* Correspondent author: Ph: 9 1 (0) 472 ll69226, 869626, 869646: Fax: 9 1 (0) 4 72 369626

Philodendrons (blue mistic, painted lady, pi nk prince, pluto, royal queen and green emperor) were selected. Top shoot cuttings ( I 0-15 em long) were collected from 2-3-year-old potted plants of the selected cultivars maintained in the germplasm collection at the institute. After defoliation , the cuttings were washed well in running tap water for 15 min before removing the leaf sheath(s) covering the axil lary bud. Stem cuttings were then immersed in 1% (v/v) labolene (Glaxo India Pvt Ltd., Mumbai) for 15 min and cleaned thoroughly. After repeated washing in tap water, noda l segments (0.8- 1.0 em long) were di ssected in a sterile air fl ow hood and the explants were surface sterilized by serial passages through 5% (v/v) sodium hypochlorite solution for 15-20 min and 0.1 % (w/v) HgCb for 5 min with a rinse in sterile tap water in between and 4-5 rinses at the end . The brownish dead ti ssue at cut ends was removed and the segments were immersed in steril e disti lied water under constant stirring for 15-20 min to remove exudates.

The different basal media tested included Nitsch9

and half and full strengt h salt and vitamins of Murashige and Skoog 10 (MS ), each supplemented with 2o/o (w/v) sucrose. After add ing the growth regulator(s) [6-benzylaminopurine (SA), kinetin (Kn), a-naphthaleneacetic ac id (NAA)] , p H of the med ia was adjusted to 5.5 and 5.8 as the case may be before adding Difco Bacto agar (0.45 %). Afrer disso lving the agar by boiling, the medium was dispensed in 15 and 50 ml aliquot into cu lture tubes (25 x 150 mm) and conical fl as ks (250 ml) and autoclaved at 121 °C and

SREE KUMAR et a!. : MORPHOGENETIC RESPONSES OF PHILODENDRON CUL TIVARS 128 1

1 x 105 Pa pressure. The cultures were incubated at 24° ± 2°C in the dark or 16 hr photoperiod provided by cool white flu orescent lamps (Philips India Ltd. , Mumbai) (40-60 JLmol. m-2 s-1

) .

After 8-12 weeks of culture initi ation in the dark, each nodal explant with shoot buds was cut vertically into 4-8 pieces, each piece having at least 2-3 buds was subcultured on hormone free medium under 16 hr photoperiod. In case, where shoot elongation/root formation was absent or not satisfactory after 6 weeks, the shoots were subj ected to a subculture for 2-3 weeks on fresh hormone free medium. Rooted as well as rootless shoots (3 -6 em long) , having 2-4 leaves, were separated, washed in tap water and transplanted in earthen pots (5 em di am.) filled with a potting mi xtu re ( I: l : 1) of ri ver sand, fa rmyard manu re and top soil. Alternati vely, for mass mul tipli cati on, nodal ex plants (0.5-0.8 em long) were excised from shoot cultures and subcul tured in the initi ation medium in the dark for 2 weeks to induce shoot proli fe ration fo llowed by ex posure to 16 hr photoperiod in hormone free medium. The shoot cuttings and rooted plants were directly transferred to the nursery or kept in a mi st chamber with 70-80% relat ive humidi ty (RH) for 3 weeks and subsequently transferred to the nursery. After 8 weeks of rearing in the nursery, the microplants were reported in pots (20 em diam.) and grown to maturity in 12-1 5 months.

The experiments were randomi zed with 3 replica­tions. Each treatment consisted of 20 replicates with one explant per cul ture vesse l. Statistical analys is was performed on the resul ts of each experiment with the computer software SPSS/PC + Vers ion 4.0 (SPSS Inc., Chicago, USA) and data were compared using ANOV A and LSD multiple range test.

Results and Discussion The nodal explants invariably released dark brown

exudates from the cut ends followed by media discolouration and blackening of the cut surface of explants in all the culti vars within two days. Ex tent of exudation was the same between culti vars and was particul arly severe in the youngest nodes. However, as in guava11 the exudates did not influence survival of the explants or their shoot forming ability. Caulogenesis was rapid when the explants were kept initi ally in the dark for two weeks and then exposed to 16 hr photoperiod. Similar observations were reported in Citrus12 and Rhododendron 13

. Among the basal nutrient medi a tes ted, high frequency of bud break occurred in Nitsch compared to half and full strength MS media (Table 1). Swelling of the nodes was foll owed by the emergence of the dormant ax il lary bud in 4-6 weeks and its growth into a single shoot in 8- 10 weeks in all the culti vars except pluto and royal queen (10-1 2 weeks) . The beneficial influence of Nitsch medium may be due to low leve l of salt espec iall y ammoniu m as reported in Anthuriwn scherzerianum 14 and Ph ilodendrons3

.

Formati on of mult iple shoots was solely dependent on the exogenous supply of a cytok inin . ln the presence of BAP, the ax ill ary bud emerged into a shoot (0.5- 1.0 em) within 2-3 weeks and addi tional shoot buds fo rmation from the base of the axi llary shoot preceded by callusing from the cut end of the ex plant was noticed after 3 weeks (Fig. I a). The requ ired concentrati on of BAP fo r the highest freq uency and number of shoot bud forma ti on varied between the cul tivars, blue mi stic, pi nk pri nce and green emperor req uired BAP (8.87 pM) while the rest required BA P (6.8 pM) for opt im um caulogenic

Fig. !- Stages of micropropagation and community rot <.!stab li shn1ent <11' Philodendron cult iva rs: (a) shoot bud formation from nodal ex plant of pink prince af'tt:r 3 wee ks or cu lture in MS mediu m supplemented with BAP (R .87 pM); (b) shoot t:longat iun of pink rrir.cc cultured after 2 weeks in MS agar basal medium ; (c) rooted pl ant s of blue mistic showing aer ial roots; (d) pink prinel.! establi shed in the community pots.

1282 INDIAN J EXP BIOL, DECEMBER 200 1

Table !- Influence of different basal media composi tion on ax illary bud emergence in six culti vars of Philodendrons.

Philodendron 1/zMS

culti vars In fec tion free Ex plants explants (%) responded

(%)

Blue mi stic 28(70) 14(50)

Painted lady 32(80) 18(56.25)

Pink prince 20(50) II (55)

Pluto 26(65) 15(57.64)

Royal queen 29(72.5) 16(55 .1 7)

Green emperor 27(67.5) I 0(37)

response (Fig. 2). Similarly the culture peri od required for initiation of shoot bud varied between the cultivars. Shoot bud initiation occurred first in painted lady (after 15 days) followed by blue mi stic (20 days), pink prince (25 days), pluto and royal queen (30 days) respectively. Similar type of variation between genotypes in respect of cytokinin requirement and period of bud initi ation has been observed in chilli 15

and tomato 16'17

. BAP was more potent than Kn (Fig. 3) in inducing the formation of shoots in all the culti vars and this was not unexpected as it is reported in other species as well 18

•19

•

Combinations of BAP and auxi ns (NAA, 2, 4-D) did not exert synergistic influence on shoot initiation (data not shown) but promoted callusing from nodal joints as well as cut ends to varied extent. The calluses so formed turned brown and degenerated during subculture in the same medium. High ratio of BAP (8 .87 JlM) and NAA (0.5 )1M) induced smal ler number of buds while concentrati on of BAP above 3.0 )1M supplemented individually or in combination with NAA inhibited bud formation altogether.

The vertically sp lit nodal explants each hav ing 2-4 proliferating buds when transferred to hormone free medium and incubated under 16 hr photoperiod st imulated shoot elongation (Fig. 1b) and development of normal leaf. Formation of new buds was absent, but 2-4 roots were initi ated in 65% of shoots within 3-5 weeks. If root formation was absent or not sati sfactory in chorous with shoot elongation, the roots were profusely formed when they were subsequently transferred to fresh hormone free medium for 2-3 weeks. It appeared that since BAP induced caulogenesis even at the lowest concentration during culture initiation , the use of the cytokinin up to 11.09 pM for optimal bud development had spill over influence and correlatively inhibited root formation. This inhi bitory effect cou ld be reversed by repeated subculture in hormone-free medium. Unlike the

MS Nitsch

In fec tion Ex plants Infection Ex plants free ex- responded free ex- responded

plants(%) (%) plants(%) (%)

29(72.5) 10(34.48) 26(65) 19(73)

3 1(77 .5) 12(38.7) 30(75) 21 (70)

24(60) 13(54. 16) 20(50) 16(80)

25(62.5) 11 (44) 2 1 (52.5) 17(8 1)

3 1 (77.5) 9(29) 24(60) 14(58.3)

29(72.5) 8(27.58) 29(72.5) II (37 .93)

earlier published report2 the addition of charcoal in the medium did not enhance root fo rmation. When the explants with buds were transferred to medium containing BAP (2.22-11.09 J1M) and incubated in light, additional bud formation was noticed but development of buds into shoots w:1s inhibited with increasing concentrations of BAP. Depress ion of shoot length by increasing BAP concentrations20 and unsuitability of BAP for uniform elongation of shoots in Philodendron2 are reported. Therefore use of hormone free medium was essential o attain normal growth of the shoot.

Concentration of agar was yet another cri ti cal factor influencing the growth of sho ts and roots. At concentrations of agar exceedi ng 0.6%, the shoot growth remained progressively stu nted and the roots got branched without elongation . The observed decline may be due to the stress exerted by hardening of the medium and insufficient avail ability of nutrients to the growing organs. Use of semi-gelled medium (0.45% agar) was beneficial for growth due to better diffusion of medium ingredients, thereby facilitating quick formation and growth of both shoots and roots. It also helped easy transplantation of the rooted plants without damaging the roots from the cu lture flask to community pots.

The roots formed in pink prince and royal queen were thin, slender and dark brown in colour whi le in other cultivars they were thick and ch lorophyllous. It was also of interest to note that certain roots of all the cultivars in culture showed negatively geotropic growth (Fig. 1c). This was not surpri sing since the vegetative habits of these plants in thei r nati ve forest habitats show that they are genetically predi sposed to growing in environmental conditions which necessitate the development of the cl imbing habi t and aerial root production2

1.22

. The rooted plantlets were uniform in all the cultivars except pink prince where definite morphological variants were obtained. The

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SREEKUMAR et al. : MORPHOGENETIC RESPONSES OF PHILODENDRON CULTIVARS

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Fig. 2- Shoot formation in noda l explants of six Philodendnm eultivars cultured in Nitsch med ium supplemen ted wit h sucrose (2 rk) . agar (0.45 %) and varied concentrations of BAP. Observations were made after 10 weeks. a: blue mi stic , b: painted lady, c: pink prince. d. pluto, e: royal quee n, f: green emperor. £3ar followeJ by the same letter(s) are not significantly different (f-'=0.05 ).

1284 INDIAN 1 EXP B IOL, DECEMBER 200 1

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Fi g. 3--Shoot formation in noda l ex plants of s ix Philodendron cu ltivars cu ltured in Nitsch medi um suppleme nted wi th sucrose 2% , agar 0.45 % and varied concentratio ns of Kn. Obse rvations were made after 10 weeks. a: blue m istic, b: painted lady, c: pink prince, d: pluto, c :

roya l queen. f: green emperor. Bar followed by the sa me letter(s) are not s igni fican tly different (?=0.05).

SREEKUMAR eta/.: MORPHOGENETIC RESPONSES OF PHILODENDRON CULTIVARS

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Fig. 4-Shoot formation in shoot culture derived nodal explants of six Philodendron cultivars in Nitsch medium containing different con­centrations of BAP. Observations were made after 8 weeks of culture. a: bl ue mistic, b: painted lady, c: pi nk prince, d: pl uto, e: royal queen, f: green emperor. Bar fo llowed by the same letter(s) are not signi ficantly different (P=0.05) .

1286 INDIAN J EXP BIOL, DECEMBER 2001

\vari ants were of three types : complete green (>20%), variegated (>60%) and albino (>20%) . Since the mother pl ants were chimeras with vari egated leaves, it was not that the regenerated pl ants got assorted into d iffe rent phenotypes. Regenerati on of genetic chimeras with and without variegation may be o f great help in deve loping new morphotypes o f horti cultural va lue.

For the purpose of multiplicati on and stocking o f shoot cultures in a ll the culti va rs, the nodal explants of in vitro shoots were subcultu red in med ium

containi ng S AP (2.22- 11 .09 J.-LM) in the dark. Caulogeni c response was rap id and pronounced and at least 30-40 shoots were formed fro m each node within 8 weeks. Altho ugh the shoot number/node showed no increase compared to the nodes o f potted pl ants used du ring culture initi ati on, the response was quick (2-4 weeks) and 100% e ffic ient and occurred at

a low level (Fig. 4) of BAP (4.44-6 .66 J-IM) . The transfer o f the nodal segments with the buds pro li ferated upo n to hormone free medium in the li ght induced rapid growth o f the buds into 5 em shoots in 8 weeks together with root fo rmati on. The multipl ication rate could be increased and a large stock o f pl ants establi shed within 2 years by repea ted subcultu ring of the nodes of ill vitro shoots .

Difference in percentage o f nursery establi shment of micropropagated pl ants between six culti vars was neg lig ible. However, s ig ni ficant di ffe rence in the frequency of establi shment was observed between hardened and unhardened shoot c uttings and rooted plantle ts. In the order o f merit the hardened rooted plantl ets showed highes t (90%) establi shment (Fig. ld) rate fo llowed by unhardened rooted (60-65%), hardened shoot cuttings (60%) and unhardened shoot cuttings (20%). These results are parti cularl y important since co mmercial laboratories direct horti cultural establi shment th rough ex vitro rooting of the shoot cuttings to reduce the cost of production. Since a 30% increase in production is possible the results suggest that in in vitro rooting foll owed by hardening is still a des irable route for success ful mass propagation o f these plants. Differences in the establishment of shoot cuttings and rooted plants between different cultivars were neg lig ible though in pink prince the a lbino pl ants perished during hardening.

Overall , the results suggest that as a lso has been reported in certain other species 7·

8 that the in vitro morphogenetic responses o f different cultivars of Philodendrons varied substantially. The protocol as

Mother plant

~ Single node (0.8-1 em)

11% Labolene (w/v) 15 min 5% Sodium hypochlorite 15-20 min 0.1% HgC12 5 min

Culture initiation

Nitsch medium + BAP (8.87 11M for blue mistic, pink prince and green emperor and 6.8 jiM for pluto, royal queen and painted lady) + 0.45% agar + 2% (w/v) sucrose, incubated in the dark for 8-12 weeks.

Shoot formation after 8 weeks Blue mistic 27 shoots/node, 2-3 em painted lady 49 shoots/node, pink prince 44 shoots/node, pluto 49 shoots/node, royal queen 36 shoots/node, green emperor

Split nodal explants vertically into 4-8 pieces each having 2-4 buds

t Subculture I

I Nitsch basal medium W 2-4 weeks incubated in light (16 hr)

Shoot elongation/rooting~ Node (0.5 em)

2-4 weeks t 0.5-2.5 em, Multiplication 4-8 nodes/shoots (Nitsch medium +

4.44-6.87 jiM SAP)

~---- 6-8 weeks ~ Subcultrue II

colmunity pot establishment (96%)

w Field establishment

Fig. 5-Protocol for rapid multiplication of Philodendron cultivars

SREEKUMAR eta/.: MORPHOGENETIC RESPONSES OF PHILODENDRON CUL TIVARS 1287

suggested in the present study for micropropagation of different cultivars can successfu ll y be used for mass multiplication (Fig. 5).

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