Indian Journal of Experimental Biology Vol. 39, October 2001, pp. 1041-1047

Cultural requirements for in vitro seed germination, protocorm growth and seedling development of densiflorum (Lam.) Schltr.

Jonojit Roy & Nirmalya Banerjee* Department of Botany, Visva-Bharati, Santiniketan 731 235, Received 10 December 2000; revised 6 June 2001

Effects of different nutrient solutions, organic supplements and growth regul ators on in vitro seed germin ation and protocorm development of Geodorum densiflorum (Lam.) Schltr. were studied. Seed germination was ve ry hi gh (up to 96%) in all the basal media, with Knudson's C and half-strength Murashige & Skoog being sli ghtly more productive than Vacin & Went. Appli cation of organic supplements and NAA had little effect on germination, but BAP proved inhibitory. After germin ation, protocorms exhibited a clear preference for peptone and NAA for much faster growth, while BAP resulted in stunted growth. Beside normal development, disorganisation of protocorms, followed by callusing occurred in presence of peptone and NAA. The calli were compact with limited growth and frequently regenerated protocorm li ke bodies. Development of seedlings was preceded by an intermediary rhizome phase. Growth of rhizomes was slow in the plant growth regulator free medium and about 15 months of culture was required for seedling formation. However, it was possible to hasten the process by 8- 10 months with the employment of NAA, which also enhanced the number of seedlings per protocorm through axi ll ary branching. Combined application of high BAP and low NAA was also useful for hi gh rate of seedling formation.

Minute seeds lacking any storage tissues, which are horticulturally important for its attractive pink to required during germination of seeds and white flowers that appear as a dense cluster in a development of seedlings, characterize the orchids. In characteristically nodding inflorescence. Today, G. nature, association with a specific fungal partner is a densiflorum is regarded as a rare species, the major 1 2 pre-requisite for orchid seed germination ' • Although cause of its decline being the uncontrolled collection 9 10 the exact role of mycorrhizal fungi is still from the wild sites • . Therefore, to prevent further questionable, it is generally considered that the damage it is extremely necessary to develop an associated endophyte provides soluble carbohydrates, efficient method for rapid in vitro propagation of this essentif\1 minerals, water, enzyme precursors and even rare and commercially important species. 3 6 hormones for germinating embryos and protocorms · . This bizarre requirement for germination has always Materials and Methods hindered rapid propagation of orchids, a group with Plant material and inoculation - The experiment enormous commercial importance, through was performed with seeds taken from seven-month conventional ex situ methods. However, the advent of old green capsules of two different . For surface asymbiotic seed germination method by Lewis sterili sation the freshly collected undehisced capsules 7 Knudson had facilitated commercial production of were first rinsed in 90% (v/v) ethanol for 60 sec, orchids considerably. Previous studies with in vitro followed by soaking in 0.1% (w/v) mercuric chloride culture of orchid seeds have shown that different solution for 20 min and finally washed thrice with species require different and often specific medium sterile distilled water. After opening the capsules with 1 3 8 composition for optimum germination and growth - · . a scalpel, about 150 seeds were inoculated in each of The present study examines the effects of three the 100 ml Erlenmeyer flasks containing 40 ml nutrient solutions, various organic additives and plant culture medium. There were 6 replicate flasks per growth regulators on seed germination and protocorm treatment, 3 for each capsule. The cultures were development of Geodorum densiflorurn (Lam.) Schltr. maintained at 25° ± 2°C under a 10-hr photoperiod Geodorum densiflorum is a teiTestrial orchid, found provided by Philips white fluorescent li ghts of 3000- in India, , some Southeast Asian countries, iux intensity. Papua and . The species is Culture media - The germination media were 11 *Correspondent author based on inorgani c salts of Knudson's C , Vacin and 1042 INDIAN J EXP BIOL, OCTOBER 2001

2 13 Wene and Murashige and Skoog , with some in a moist and shady place in the experimental garden modifications. The modified Murashige and Skoog for about two months. After commencement of new (HMS) medium consisted of half-strength macro and growth the seedlings were then shifted in their natural micro salts (iron-EDTA in full strength) and 100 mg/1 habitat during rainy season to prevent possible myo-inositol. In both Knudson's C (KC) and Yacin desiccation. and Went (VW) media, the original iron source was replaced with iron-EDTA as in the HMS. In all media Results and Discussions 2% (w/v) sucrose served as carbon source. In Seed germination-Germination of seeds began addition, different organic additives and plant growth after about 4 weeks of inoculation. The initial stages regulators (PGRs) were supplemented to each type of 1 of germination were typical of most orchids , medium in various combinations (Table 1). The involving swelling of the embryo, followed by vitamin mixture consisted of 1 mg/1 nicotinic acid, 1 rupturing of the seed testa and subsequent emergence mg/1 pyridoxine HCI and 10 mg/1 thiamine HCI. The of the protocorm (Fig.1a). The results of different pH of the media was adjusted to 5.2. The media were treatments, shown in Table 1, have clearly solidified with 0.9% (w/v) agar and autoclaved at 1.02 demonstrated that G. densijlorum have no stringent kgcm·2 for 20 min . nutritional requirements for initiation of germination, Germination and protocorm growth- The cultures unlike many other tenestrial orchids of temperate were examined every week for initiation of region that generally show preferences for low salt germination. Emergence of the embryo from the seed 3 media, in which the form of nitrogen also appears to testa was considered as germination . Growth of 3 8 15 16 be vital for germination · · · . The use of all 3 types protocorms is expressed in terms of protocorm length, of germination media, differing in the total nitrogen for which 50 randomly selected samples were level as well as in the ratio of NH/ and N03· , resulted measured for each treatment. The results of different in high frequencies of germination, with KC and morphogenetic responses were taken after 3 months HMS being slightly more productive than YW. of culture. Modifications of these · media with various organic Protocorm culture - After 3 months of initial supplements had little effect on germination; onl y the culture the protocorms from HMS-CwPVm treatment employment of peptone in VW medium improved (Table I) were subcultured for further growth and germination to some extent. development. For this purpose the same medium was The use of BAP, with or without additional NAA, used as the PGR-free control, along with three PGR­ was inhibitory to seed germination and lowered its treatments, consisting of 1 mg/1 NAA, combinations frequency significantly. Regarding germination of of 2 mg/1 BAP and 1 mg/1 NAA and 4 mg/1 BAP and orchid seeds in presence of exogenous cytokinin, 3 1 mg/1 NAA. Data regarding rhizome growth and types of response have been recorded previously - seedling formation in the PGR-treatments were taken either germination is improved or inhibited, or there is 3 17 after 3 months of culture. However, seedling no effect ' . Orchid seeds that do not require development in the control was extremely slow and exogenous cytokinin for germination are known as required additional period of subcultures (performed cytokinin autonomous, since they contain sufficient 3 18 at 3-month intervals) and hence the results of this endogenous cytokinin • . The requirement of treatment were collected after 12 months of culture. cytokinin for germination is considered related to the Statistical analysis- The mean values of different utilization of lipids that constitute primary storage morphogenetic responses were analysed by ANOV A material in most orchid seeds and it has been observed (analysis of variance), in conjunction with Duncan's that unless storage lipid is utilized germination does 3 19 Multiple Range Test (a = 0.05). The experimental not continue · . In many terrestrial species, e.g. units were assigned to 'randomized complete block Cypripedium reginae, C. calceolus and C. candidum, design', with single replicate per block. All statistical a definite cytokinin preference for germination and 3 16 20 analyses were performed according to Little and protocorm growth has been reported ' ' • In all these 14 Hills • species a low level of cytokinin has been found to be Hardening of seedlings - Initially 30 well-rooted optimum. Although the inhibitory effect of BAP in G. seedlings, each with a small segment of attached densijlorum could be due to its particular level of rhizome, were transferred to clay pots, containing a application, it is apparent that this species does not mixture of garden loam and sand in 2: I ratio and kept require any exogenous cytokinin for germination, as ROY & BANERJEE: IN VITRO PROPAGATION OF GEODORUM DENSJFLORUM 1043 evident from the high frequency of germination (upto most of the treatments the protocorms remained more 98%) in its absence. In contrast to BAP, the use of or less unchanged with the leaf primordia occurring as NAA by itself had little impact on germination . scale-like leaves. However, among the various percentage. However, in KC medium its presence treatments significant differences in the size of suppressed the inhibitory effect of BAP significantly. protocorms were noted (Table 1). The effect of Previously, a similar type of interaction between coconut water by itself was inconsistent and proved auxin and cytokinin has been reported in seed culture beneficial only in KC medium, but the application of of Cypripedium reginae, where auxin could peptone exhibited a growth promoting effect in all the counteract the inhibitory effect of supra-optimal media. Addition of coconut water and vitamins along 20 cytokinin on seed germination . with peptone showed no further growth improvement. Morphology and development of protocorms - In The presence of NAA, along with these organic the early stage of growth, the protocorms appeared as supplements improved the growth even further, while translucent white bodies with a pointed basal end. BAP proved inhibitory. The growth response of After passing through an initial phase of swelling, a protocorms was intermediary in the combined number of tiny leaf primordia emerged from the . presence of these two PGRs. apical part of the protocorm (Fig.l b), while clusters of In general, reports of peptone usage in orchid tubular rhizoids developed from the basal region. culture media are less common and its effects are not 21 22 Soon there was enhancement of protocorm size, yet well established ' • Still, its application in a associated with slight elongation of the leaf primordia number of species has shown notable improvement of 23 27 (Fig.lc). At this stage the colour of the protocorms germination and growth '· · • The analysis of this ranged between whitish, light yellow and green. In substance has shown that it could be a major source of

Table !- Effects of different treatments on seed germination and protocorm development of Geodorum densijlorum. Treatment Frequency of Protocorm death Protocorm length± SE Callusin g Shoot growth germination± SE (%)* (mm)* (%)* (%) (%)* KC-B 96.3 :;!:0.17 nbc 1.44 :;1::0.05 hi 0.2 e KC-Cw 98.0 :;!:0.17 ab 1.4 d 1.9 :;!:0.09 fg 3.3 e KC-P 96.5 :;!:0.18 nbc 2.2 d 2.17 :;!:0 .08 cf 16.1 bed

KC-CwPVm 97.7 :;!:0.08 ab 0.8 d 2.24 :;!:0 .1 3 ef J3 .J bcd KC-CwPVm (N I) 97.6 :;!:0.05 ab 13 .\c 3.18:;t0 .1 6b 19.9 be KC-CwPVm (Bpi) 81.7:;!:1.52 h 0.91 :;t0.02j

KC-CwPVm (Nl+Bp2) 85.9 :;!:0.03 g 2.21 :;!:0.1 cf 0.7° 11.4 VW-B 92.9:;1::0.96d 1.27 :;!:0.04 ij VW-Cw 94.7 :;1::0.31 bed 1.32 :;!:0.04 hi VW-P 96.5 :;!:0.47 abc 2.48 :;!:0.13 de !4.4 bed VW-CwPVm 98.4 :;!:0.73 a 2.95 :;!:0.16 be 12.3 cd VW-CwPVm (Nl) 96.8:;!: 1.56 abc 3.3:;!:0.16nb 31.8 n VW-CwPVm (Bpi) 87.3:;!:0 .1 8fg 1.09 :;!:0.04 ij

VW-CwPVm (Nl+Bp2) 89.6 :;!:1.48 ef 2.09:;!:0.17 [ 1.5 e 9.8 HMS-B 96.8 :;!:0.0 I abc 1.45 :;!:0.04 hi HMS-Cw 96.4 :;1::0.21 nbc 1.67 :;!:0.12 gh HMS-P 97 .3 :;!:0.32 abc 2.78:;!:0.14cd 20.9b HMS-CwPVm 96.() :;!:0.41 nbc 1.9d 3.0:;!:0.14bc 10.3 de HMS-CwPVm (Nl) 95.0 :;!:0.67 nbcd 18.1 b 3.62 :;!:0.2 a 13.8 bed HMS-CwPVm (Bpi) 92.1 :;1::0.1 de 1.09 :;!:0.35 ij 2.9 HMS-CwPVm (Nl+Bp2) 92.2 :;1::2.65 de 2.2:;t0.16cf 0.8 c 3.2 KC = Knudson's C, VW = Vacin & Went, HMS = half-strength Murashige & Skoog, B = Basal medium, Bp = BAP (mg/1), Cw = Coconut water (10%, v/v), N = NAA (mg/1), P =Peptone (0.2%, w/v), Vm =Vitamin mixture. *Mean values followed by same letter are not significantly different at 0.05 level (Duncan's Multiple Range Test). 1044 INDIAN J EXP B!OL, OCTOBER 200 1

Fig. !-Seed germination, protocorm development and different morpho-types: Ia. Emergence of protoconn (-+) from seed coat. Bar = 0.4 mm; lb. Development of leaf primordia(-+) in protocorm. Bar= 0.4 mm; le. Elongation of leaf primordia (-+). Bar = 0.4 mm; ld. Most common morpho-type of protocorm. Bar= 1.0 mm; I e. Protoconn with two shoot apices (-+ ). Bar= 1.0 mm; I f. Protocorm with sma ll protrusions (-+ ). Bar = 0.4 mm: I g. Elongated protoconn . Bar = 1.0 mm; I h. Rhi zome (-+) fom1ation in protocorm, followed by shoot develo pm ent( ~ ) . Bar= 2.0 mm. ROY & BANERJEE: IN VITRO PROPAGATION OF GEODORUM DENS/FLORUM 1045 organic nitrogen, comprising primarily various amino (Table 1; Fig.l h). Shoot development of this kind was 8 21 28 acids · · . Therefore, the growth promoting effect also noted in presence of BAP alone (in HMS observed in G. densiflorum could be due to the supply medium), though the protocorms were not elongated, of ample exogenous amino acids present in the they had normal morphology. In the second morpho­ peptone. It is possible that in the early stages of type, the protocorms had 2-3 shoot apices (Fig.le), protocorm development the endogenous biosynthesis while in the third type a large number of pointed of amino acids may not be adequate for healthy and protrusions were detected on the surface of the faster growth. However, due to its complex nature the protocorms (Fig.l f). These last two abnormal effect of peptone on orchid seed culture would always morpho-types were present in most of the treatments be difficult to interpret. Even so, the application of in very small percentage (less than 1 %). this substance could be of immense significance in the In a number of treatments, the protocorms after an commercial production of this species. initial period of growth exhibited a process of Apart from the common protocorm morphology disorganization and subsequent callusing (Fig.2; (Fig.l d), three other morpho-types were also Table 1). The resultant calli were hard, white to recognized among the treatments. In the first type, yellow in colour and had limited growth. which was found in the combined presence of BAP Regeneration of protocorm like bodies (PLBs) and NAA, th e protocorms showed an elongated occurred frequently (about 60 to 70%) from these appearance from the very early stages of development calli (Fig.3). The number of PLBs per callus body (Fig.lg). In the later stages of growth, a small ranged between 5 and 20, each capable of producing percentage of these elongated protocorms developed rhizome upon subculturing. The frequency of leafy shoots, which was preceded by a short rhizome callusing was particularly high in presence of

Fig. 2- A callus body developed through disorganisation o r protoeonn. Bar= 1.0 mm ; Fig.3. Regeneration of PLBs (->) from callus. Bar = 1.0 mm; Fig.4. A cluster of rhizomes branches (->)showin g shoot development(~), followed by rooting (<==>) in presence of NAA. Bar = 5.0 mm. 1046 INDIAN J EXP BIOL, OCTOBER 200 1

peptone. The application of NAA and BAP had death considerably. Hence, it appears more plausible contrasting effects on frequency of callusing. In VW that the increased mortality rate is a consequence of medium NAA increased the frequency of callusing nutrient depletion in culture media caused by fast significantly, up to about 32%. The combined growing protocorms, a suggestion similar to that 29 application of BAP and NAA in all the media lowered made by Stoutamire . In th at case the loss of the percentage of callusing, while BAP alone protocorms could be reduced by less dense completely prevented disorganization of protocorms. inoculation of seeds or by frequent subculturing. Although these protocorm-derived calli showed Development of seedlings - The result of the hardly any growth, their potentiality to regenerate present study has shown that development of rhizome PLBs could be useful to develop in future a callus from the protocorm is a pre-requisite for seedling associated micropropagation system for this species. formation in G. densiflorum. Previously only few Gradual browning and eventual death of orchids, particularly different species of terrestrial Cymbidium, have shown this type of morphogenetic protocorms was observed in a number of treatments in 30 32 varying frequency (Table 1). In most cases protocorm development . . Although most rapid seedling necrosis was quite low, but the application of NAA development occurred in germination media enhanced it considerably, up to about 29% in VW supplemented with combinations of 2 mg/1 BAP and 1 medium. In general, necrosis of developing mg/1 NAA, the yield was very low, as mentioned protocorms is a common phenomenon in orchid seed previously. However, considerable improvement was culture and there are different opinions regarding this. achieved by the application of same combination of Harvais20 observed that protocorm necrosis in PGRs during later stages of protocorm development. Cypripedium reginae is associated with the Shoot formation was best in combined presence of 4 production of excess phenolics, which is possibly due mg/1 BAP and 1 mg/1 NAA (77.8%), followed by 2 to increased activity of polyphenol oxidase and mg/1 BAP and 1 mg/1 NAA (55.6%). In the PGR-free catalase activity triggered by certain cultural medium rhizomes showed extremely slow barowth , conditions. While Stoutamire29 reasoned that occasional ax illary branching (about 6%) and improper nutrients and lack of essential growth conversion of rhizome tip into leafy shoot occuned stimulating substances in the culture media may result after 11-12 months of subculture. Shoot formation in protocorm mortality. Still in other study, De Pauw was followed by swelling of the basal portion to form et al. 3 suggested that in the seed culture of a pseudobulb and later by root formation. Still, the Cypripedium candidum the 'necrotic protocorms' frequency of such seedling differentiation was very could actually be the non-viable embryos that enlarge low (37.5%) in this medium. In this regard, and break through the seed testa following imbibition application of NAA during the protocorm culture was of water. However, this last postulation could be significant, which resulted in vigorous growth of overruled for G. densiflorum, since in a number of rhizomes and hastened the process of seedling treatments, especially in the basal media and HAP­ formation by about 8-10 months, as weli as increased treatments, there was complete absence of protocorm its frequency (50%). The number of seedlings death. In addition to this, majority of the necrotic obtained per protocorm was also enhanced by NAA protocorms were of considerable size, unlikely to be (Fig.4) through stimulation of axillary branching attained by simple imbibition, and many were found (72% ). The mean number of axillary branches was with distinct leaf primordia. In a previous study about 6 per protocorm. In contrast to control and auxin-induced protocorm mortality has been reported NAA-treatment, rooting of newly formed shoots was 20 absent in combined presence of BAP and NAA and in Cypripedium reginae . Therefore, it is possible that in G. densiflorum the high rate of death in NAA­ needed an additional PGR-free subculture for treatments is a direct toxic effect of this auxin. On the development of rooted seedlings. This is most likely due to the inhibitory effect of the cytokinin, as was other hand, comparison of growth and death rates of 33 34 different treatments shows that maximum death found in many other angiospermic plants · . occuned in media where growth of protocorms was Ex vitro establishment of seedlings-In the most vigorous and least or absent in the slow growing hardening process survival rate of seedlings was about cultures. For example, addition of BAP along with 87%. Initiation of new growth occurred through NAA not only suppressed the growth promoting development of an axillary shoot that later formed a effect of the latter, but also averted NAA-induced daughter pseudobulb, while the parent pseudobulb ROY & BANERJEE: IN VITRO PROPAGATION OF GEODORUM DENSIFLORUM 1047 remained as a green back-bulb with withered shoot 17 Arditti J & Ernst R, Physiology of orchid seed germination, region. These plants were then successfully in Orchid biology: Reviews and perspectives, Jll, edited by 1 Arditti (Cornell University Press, Ithaca, New York) 1984, transferred in the wild site, with a 60% survival rate 176. after 26 months. 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