Nov 9, 1998 - been observed in our earlier observations with. Chlorophytum borivilianum~ and C. orchioides. 1A . Leaf explants are most suitable material to ...
Indian Journal of Experimental Biology Vol. 38, February 2000, pp. 145-148
A method for large-scale multiplication of Curculigo orchioides through bulbil formation from leaf explant in shake flask culture Sarabjeet S Suri, Dilip K. Arora & Kishan G Ramawat Laboratory of Bio-Molecular Technology, Department of Botany, M L Sukhadia University, Udaipur J I J 00 I , India Received 9 November 1998; revised 28 October 1999
An efficient method has been developed for large-scale multiplication of Curcllligo' orcizioides (Hypoxidaceae), an endangered medicinal plant, through direct bulbil formation from leaf explants in shake flask cultures. Leaf-segments (7xl0 rnm) were cultured in Bs liquid medium containing KNO, (200 mgNL·'J. (NH4hS04 (50 mgNL- 1), benzyl adenosine (2.2 ~, adenine (0.11 mM), indole butyric acid (1.0 ~M) and polyvinyl pyrrolidone (250 mgL-\ About 95 % explants produced maximum number of bulbils (S46/tlask at 6 weeks growth) in the medium. Shake flask cultures yielded 2737 bulbilslL medium whereas static cultures yielded 624 bulbilslL medium. Germination of bulbils was maximum (90.62%) on agar-gelled B5 medium containing benzyl adenosine (2.2 ~ and gibberellic acid (3.5 ~ . Plantlets developed in vitro were successfully transferred to soil with a high rate of survivability (90%) and were comparable to natural population in growth and vigour.
Tuberous roots of Curculigo orchioides (Hypoxidaceae) are widely used as tonic for health, vigour and vitality due to the presence of flavanone glycosides. The plant is naturally propagated through seeds and underground bulbils. Over-exploitation of the plant associated with poor seed set and germination has made it an endangered species I . Efforts are needed to propagate this species on the commercial scale to meet the demand of pharmaceutical firms and to protect the existing natural population of the species. Plant tissue culture technique has become a powerful tool to develop 2 micropropagation methods for such plants . Earlier, we reported shoot proliferation from leaf and stemdisc explants, bulbi I formation from stem-discderived callus), somatic embryogenesis and bulbils formation from leaf explants in C. orchioides4 . Excised leaf ex plants from in vitro plantlets serve as aseptic reservoir of inoculum required for large-scale multiplication. Direct regeneration of embryos and bulbils from the leaf explants makes them an excellent system for such a purpose. Bulbil formation has gained considerable attention as a novel method for micropropagation due to easy in transportation, better survivability of germinated bulbils in soil, low cost, and rapid propagations. In this communication, we report a method for large-scale propagation of Curculigo orchioides
through direct bulbil formation from leaf explants in shake flask culture.
Material and Methods Young leaves of C. orchioides from asepticall y maintained plantlets, raised through shoot 3 proliferation from stem-disc and leaf explants were used as source material. Leaf segments (7 x 10 mm) were aseptically transferred in liquid media selected on the basis of our earlier results. Media used wereMedium I: Bs medium 6 containing KNO, (170 mgNL- 1), (NH 4hS04 (85 mgNL' \ 6-benzyl adenosine (BA, 2.2 !lMY, adenine (0.1 I mM) , 2,4 ,5trichlorophenoxyacetic acid (2,4,5- T, 1.0 ~lM) and polyvinyl pyrrolidone (PVP, 250 mgL-1); Medium II: Bs medium contammg KNO., (200 mgNr\ 1 (NH 4hS04 (50 mgNL· ), BA (2.2 !lM), adenine (0.11 mM), indole butyric acid (IBA, 1.0 !lM) and PVP (250 mgrl); and Medium III: Murashige and Skoog (MS) medium 7 containing BA (2.2 !lM) , adenine (0.11 mM), IBA (1.0 !lM) and PVP (250 mgL· 1 ). The medium was adjusted to pH 5.8 with 0.5 N Hel or NaOH after incorporation of all ingredients of the medium and 200 mL of medium was dispensed in each conical flask (1000 mL, Borosil). Flasks were closed with non-absorbent cotton and autoelaved for 20 min at 121 °C at 1.05 kg em- 2 . Seventy five explants were transferred in each flask and three such
INDIAN J. EXP. BIOL. , FEBRUARY 2000
146
replicate cultures were used for each treatment. Cultures were incubated at 26°±0.5°C under white fluorescent light (Philips cool TL 36W/54, 220 volts) with a total irradiance of 36 /J- mol m· 2 S·I for 16 hr photo-period and 55-60% RH. Cultures were agitated on a rotary shaker at 70 rpm (with 5 cm displacement from the central axis) and medium was replaced with fresh medium every 2 weeks to remove leached dark brown substances. Efficiency of two different culture systems viz., agar 4 gelled static cultures and shake flask cultures, was compared for production of plantlets. Bulbils produced in vitro (20-150 mg fresh wt) were transferred on to B5 medium containing different concentrations and combinations of BA (1.1-2.2 !J-M) and GA3 (3.5-17.5 /J-M). These media were gelled with 0.8% (w/v) bacteriological agar (BDH, England) and 50 mL of each medium was poured in separate conical flasks (250 mL, Borosil). Plantlets developed in vitro through germinated bulbils were washed with a systemic fungicide, carbendazim (2-methoxy carbamoyl)-benzimidazole (1 %, w/v) for 5 min and planted in plastic pots (150 mL) containing sterilised garden soil: composed (I: I) mixture. To maintain high ambient humidity (RH 7080 %), pots were covered with transparent polythene bags containing a few pores to allow gaseous exchange. Plants were irrigated as and when required (25 mL/pot, tap water). After 15 days growth in soil, polythene bags were removed and plants were exposed to sunlight 2 hr/day and for rest of the period, plants were kept in moisture and shady place. Onemonth-old plantlets were finally transferred in the field, exposed to external environment.
Results and Discussion Leaf-segments (7x 10 mm) transferred in MediumI, II and III produced varied number of bulbils without intervening callus (Table I) . Bulbi Is were initiated after 15 days of inoculum . Maximum number of bulbils (3501flask after 4 weeks growth and 5461f1ask after 6 weeks growth) were produced in the Medium-II. Explant response in this medium was 94.6 %. No further increase in the growth of bulbils was recorded after 6 weeks in all the media tested. Incorporation of PVP (250 mg L· I ) in the mediulll reduced leaching albeit to lesser extent. Bulbi Is developed in vitro were brown in colour with brownblack crust and a dormant shoot apex (Fig. I). Ungerminated bulbils vary in their size from 5 to 100 mg with majority of bulbils (31%) weighing up to 10 mg (Fig. 2). Productivity of two different culture systems vir., agar gelled static cultures 4 and shake flask cultures, containing the optimal medium, was compared (Table 2). Explant response in shake flask cultures was though lower (94.6 %) than that in agar-ge lled static cultures (100%), but bulbils yield was much higher. Shake flask cultures produced 2737 bulbils per litre medium whereas static cultures could produce only 624 bulbils per litre medium at 6 weeks . Thus, clearly indicating the superiority of shake flask cultures over static cultures in producing higher number of bulbils by accommodation of higher number of explants per litre of the medium. This yield perhaps can be increased further by manipulating the size of the leaf· explants. Though the number of bulbils per explant remained lower in the liquid medium than static medium, over-all yield per litre of
Table I-Effect of different media on bulbil formation [Values are mean of 3 replications] Shakeflask medium
1
"
III
Expl ant respon se (%)
90.6 94.6 93.3
Growth (4 weeks) Bulbils/ explant Bulbils yield 3.3
4.7 1.4
Growth (6 weeks) Bulbils/expl ant Bulbils yield
250 350 108
3.5 7.3 2.5
266 546 IX7
Table 2--Yield of bulbils in static and liquid medi a. Culture vessels Static flask** (250 mL) Shake flask ( I 000 mL)
Medium*/ fla sk (m!)
50 200
Explants/ flask
3 75
(% Explant response)
Bulbils/explant
Bulbil yield aftcr 6 weeks growth (L· 1 mediulll)
10.41 (100.0) 7.30 (94.6)
2737.50
624.60
*8 5 medium containing KNO, (200 mgNL· 1) , (NH 4hS04 (50 mgNL 1), BA (2.2 J..lM), adenine (0.11 IllM ), IBA ( 1.0 pM) and PYP (250 IllgJ"I). **Data from our previous results4.
SURI eta/.: A METHODF OR LARGE-SCALE MULTIPLICATION OF CURCULICO
medium was significantly higher as liquid medium contained higher number of explants. Isolated in vitro produced bulbils developed into complete plantlets when transferred on to B5 medium containing different concentrations and combinations of BA (1.1-2.2 11M) with or without GA3 (3.5-17.5 11M). High percentage germination was observed in the bulbi Is weighing 41-80 mg. It is presumed that small sized bulbils were not fully matured while oversized (~ 100 mg) bulbils developed certain level of dormancy (Fig. 3). Germination of bulbils was maximum (90.62% after 20-days growth) on the medium supplementeq with BA (2.2 11M) and GA3 (3.5 11M, Table 3, Fig. 4). Higher concentrations of GA, (17 .5 11M) in combination with BA inhihited
.. -
.'!
~
ORCHIOIDI~S
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bulbil germination. Vigour of the plantlets developed through germination of bulbils was optimal in the medium containing BA (2 .2 11M) and GA ~ (3.5 !-1M). Plantlets developed in vitro were successfully transferred in soil with high survival rate (90%) and plants were comparable to natural population in growth and vigour. Preserice of storage organ (bulbil) with th e saplings increased their field survivability. This has been observed in our earlier observations with 1A Chlorophytum borivilianum~ and C. orchioides . Leaf explants are most suitable material to establish 1 in vitro cultures and for rapid multiplication . Leaf explants provided contamination-free cultures which is, otherwi se, a major problem 111 culturin g underground stem-disc as a source of explant or for large inoculum required for bioreactors. Rate of multiplication and survivability of in vitro raised plantlets in soil was high in C. orchioides. Bulbil formation from leaf explants in shake flask cultures without intervening callus can be efficiently used to preserve true to type traits in propagules and for rapid multiplication. Bulbil formation has gained considerable attention as a nove l method for micropropagation due to ease of handling, storage, Table 3-Effect of different concentratiolls and comhinations or BA and GA, on the germinatioll or bulhils [Values are mean or 40 bulhil s l GA3 (11M)
% germination
Initiation time (days)
Lear le ngth (cm)
1.10
0 3.50 17.50
58.12 81.87 61.25
20 15 20
2.5±0.3 3.5±O.6 2.0±O.4
2.20
0 3.50 17.50
86.25 90.62 60.00
15 12 20
4.2±O.5 5. 1±1.1 2.0±O.6
BA (/lM)
Fig. I-Production of bulbils from leaf-segment explants of C. orchioides in skake flask culture. Fig. 4-GerminatioQ of bulbils on B5 medium supplemented with BA (2.2 11M) and GAJ (3.5 11M ).
-_.. _-.----------1
40 CD Cl
-e
30
co
.,
~20 CD
(l.
Bulbils
10
o 1-10
II I
.L___J
41-50 Fresh weight of bulbils (mg)
91-100
Fig. 2-Variation in the fresh weight of ungerminated bulbils.
o
41-.:
