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In vitro organogenesis and somatic embryogenesis in Adenia hondala (Gaertn.) de Wilde, an endangered medicinal plant of the Western Ghats.

Introduction

Adenia hondala (Gaertn.) de Wilde. Is an endangered species of potential medicinal value. It has been red listed as vulnerable and rare in South India [1]. It is endangered in Tamilnadu [2].

The major hindrance for conventional propagation of Adenia hondala is its poor seed set and seed germination. Adenia hondala is a large perennial climbing herb belonging to the family Passifloraceae. It is found in the forests of Western Ghats and other parts of Peninsular India. The roots are large, tuberous, the leaves are membraneous, cordate and deeply lobed. Flowers are rather large and monoecious. The fruits are 7 cms in diameter and poisonous. Seeds are discoid and arillate. The herb is reported to posses antiseptic properties. The juice is used as medicine in Sri Lanka. They are used as a pectoral. The roots are poisonous and are used in skin troubles [3]. The tuber powder is used to treat expectorant cough and hoarness of voice. It is also used to increase lactation in nursing mother. The juice of tuber is used to cure intermittent fever [4].

Seeds are not readily available and therefore vegetative material is preferred as the source of propagation by in vitro techniques. Thus a wider genetic base can be maintained.

Considering the importance of the plant, and the threat faced by them, the main objective of the present investigation was to establish an efficient and easily reproducible protocol for large scale clonal propagation through multiple shoot induction and somatic embryogenesis through different pathways. The development of a tissue culture methodology can provide an alternate means for the mass production of Adenia hondala to satisfy the demand in the market and also for reintroduction programme for conservation.

Biochemical procedures were adopted to quantify the metabolities in in vitro systems, which could provide an insight into the metabolic changes occurring during the morphological stages in the development.

Materials and methods

One-year-old plants of Adenia hondala grown and maintained in the green house, Department of Botany, Sacred Heart College, Thevara, Kochi were used as the source of explant. In vitro propagation protocols were carried out through standard techniques. Excised shoot tips, nodal segments (3-4cms), tender leaves (2nd-3rd) from the apex and internodal segments (2-3cms) were used as explants. The explants were surface sterilized with 0.1 % (w/v) Hg[Cl.sub.2] for 6 minutes and rinsed thoroughly with sterile distilled water. Surface sterilized explants were cut into small pieces and placed on media containing [MS.sup.5] basal mineral salts, 3 % (w/v) sucrose, 0.8 % agar and 100 mg [1.sup.-1] meso-inositol.

Plant growth regulators namely 2, 4-D, NAA, IAA, BAP, Kn and 2iP were used in different combinations and concentrations for callus induction, plant regeneration and somatic embryogenesis. The individual shoots were excised from shoot clumps and transferred to half and quarter strength and auxin containing MS medium for rooting. The pH of medium was adjusted to 5.8 and autoclaved at 121 [degrees]C for 20 minutes at 15 lbs. All cultures were maintained at 25 [+ or -] 2 [degrees]C with a photoperiod of 16/8 hr light/dark cycle. The culture room was equipped with white fluorescent lamps with a light intensity of 2000 lux. The effect of additives supplementation on regeneration was also studied. The additives tried were CAP (0.5 - 2.5 mg [1.sup.-1),] Biotin (0.5 - 2.5 mg [1.sup.-1]), [GA.sup.3] (0.5 - 2.5 mg[1.sup.-1]), ABA (0.5 - 2.5 mg [1.sup.-1]) and TDZ (0.5 - 2.5mg[1.sup.-1]), CH (50 - 500 mg [1.sup.-1]).

The plantlets with well-developed roots were transferred into pots containing sterile Soilrite. The pots were maintained in a polythene chamber in the culture room and irrigated with 10 % MS solution once in a week. After one month the plantlets were transferred to the field or transplanted to earthen pots for further growth in the green house.

For each treatment, minimum of 20 replicates were used and all experiments were repeated thrice. The mean and standard error of the number of shoots produced per explant were calculated from the replicates for each treatment. The results were analyzed statistically using the analysis of variance.

The histological screening of cultures was carried out at uniform time intervals following conventional histological techniques [6].

The total proteins [7] were estimated in trichloroacetic acid precipitates [8], which had been treated, to remove contaminants. Bovine serum albumin (Sigma) was used as standard. All determinations were carried out 3 times to permit standard evaluation.

For the qualitative estimations of starch and soluble sugar, the samples were collected at 10 days intervals and 100 mg dry weight samples were extracted with 5 ml methanol. This was extracted once again and the combined extract was reduced to dryness. An aliquot (0.5ml) of this solution was employed for the determination of soluble sugars by anthrone colorimetric method [9]. Then starch was extracted from the residue in boiling distilled water and determined using 12 K[I.sup.9].

Results and discussion

The full strength MS medium, with higher levels of inorganic nitrogen was selected as the basal medium. The nodal explants exhibited bud break within a week. 3rd to 6th nodal explants responded better in terms of bud elongation. Shoot tips and internodal explants did not induce caulogenic response. Younger nodes close to the shoot tips are under the inhibitory influence of some factors or other released from the shoot tip [10]. The preference of nodal explants over the apical dominant shoot tips is well documented in Fagus [11] and Chestnut[12].

The nodal explants produced 4.25 [+ or -] 0.95 shoots / culture in days at 0.5 mg [1.sup.-1] BAP. In many plant species, cytokinins play an important role in overcoming the influence of apical dominance and in enhancing branching of lateral buds [13]. The nodal explants produced the maximum number of shoots in BAP and Kn combination. More multiple shoots were induced at low concentrations. BAP and Kn have synergistic effects in promoting shoot initiation [14]. Increase in the concentration of cytokinin supporessed bud break, decreased the number of shoots and retarded shoot elongation. This is in consonance with the findings in Ocimum species [15].

At an optimum combination of 0.5 mg [1.sup.-1] BAP + 0.5 mg [1.sub.-1] Kn + 10 % CW, 8.25 [+ or -] 0.95 shoots/culture were obtained. Addition of coconut water (CW) improved the results (Fig 1a, Table 1). Significant increase in shoot length was noticed when 0.5 mg [1.sup.-1] 2iP was combined with BAP, where the shoots elongated to a height of 8.25 [+ or -] 0.6 cms with in 6 days.

The source of explant and growth regulator combination used in callogenesis exhibited significant influence on organogenesis. At 0.25 mg [1.sup.-1] BAP + 1.5 mg [1.sup.-1] NAA optimum callusing was obtained from internodal explants. The green compact calli derived from internodal explants showed highest shoot morphogenesis within 5 - 10 days when sub cultured into BAP and Kn combinations. An average of 14.25 [+ or -] 1.5 shoots / culture was produced at an optimum concentration of 1 mg [1.sup.-1] BAP + 0.5 mg [1.sup.-1] Kn + 10 % CW (Fig. Ib, Table 1). When the medium was refreshed after 30 days, further bud break was observed. The shoots were not developed in synchrony (Fig. 1c).

The elongated shoots of 5 - 8 cms, were transferred to different strength MS for root induction 20.75 [+ or -] 4.34 roots / shoot were produced in half MS medium devoid of plant growth regulators. The roots produced were long, with well developed root hairs and branches (Fig. 1d). Shoots administered with auxins induced a few short, slender roots without root haris. Extensive callusing at the base was also observed when auxins were used for rooting. Although auxins are commonly used for rooting, it is not required in most cases [16]. Half strength MS medium without growth regulators induced more roots compared to full strength MS in Gladiolus hybridus [17] and in Celastrus paniculatus [18]. Low ionic strength medium is frequently used for in vitro rooting. This may be due to the need for only small amount of total nitrogen for rooting [19].

In Adenia hondala somatic embrygenesis was induced directly from the leaf explants and indirectly from the internodal derived callus. The highest number of embryos was obtained in medium supplemented with NAA and BAP. The combination of NAA and BAP was necessary to induce somatic embryogenesis in leaves from young plants of Quercus suber also [20]. The highest mean number of direct somatic embryos 34 [+ or -] 0.2 were induced in 45 days from leaf explants. The inclusion of CH (200 mg [1.sup.-1]) with 2 mg [1.sup.-1] NAA and 0.5 mg [1.sup.-1] BAP significantly enhanced the percentage of initiation and number of embryoids from leaf explants (Fig 1e, Table 2).

Indirect embryogenesis was observed in green compact calli obtained from internodes at a combination of 1 mg [1.sup.-1] NAA + 0.5 mg [1.sup.-1]BAP. Within 25 days of culture 59.75 [+ or -] 4.5 embryos were produced. The embryo induction further increased to 62.4 [+ or -] 2.5 embryos when 0.5mg [1.sup.-1] TDZ was combined. Higher concentration of TDZ above 1 mg [1.sup.-1] reduced the frequency and the number of embryos.

For embryo maturation, embryoids were transferred to maturation medium containing 6 % sucrose. Maturation of the embryos and plant development generally require a culture medium containing cytokinin and a low concentration or no auxin [21]. Sucrose played a vital role in the maturation of embryos. The medium containing 6 % sucrose gave better response in early stages if embryo development and enhanced embryo maturation. The high osmotic potential of MS media containing high concentration of sucrose fostered embryo development and maturation [22]. 25.3 [+ or -] 1.8 embryos matured in 34 days when 6 % sucrose along with 0.05 mg [1.sup.-1] NAA + 1 [mg.sup.-1] BAP + 1.5 mg [1.sup.-1][GA.sub.3.] Lower and higher concentration of sucrose levels in the medium reduced embryo maturation. After 15 days of culture globular embryos became heart shaped and then torpedo shaped. The embryos elongated and showed distinct bipolar growth forming a shoot pole and a root pole (Fig 1, g, h).

Rooted plantlets were acclimatized gradually to low relative humidity conditions. The plantlets showed higher percentage of survival when transplanted in soilrite. Normal growth of potted plants was observed after transfer to the field conditions with 90 % survival (Fig 1, i).

Despite the fact that plant cells display a remarkable potential for cellular totipotency, behaviour of plant cells or explants in tissue culture medium is unpredictable. It is assumed that differentiated plant cells retain their ability to revert to embryogenic condition and generate a complete new plant through organogenesis or somatic embryogenesis. Differentiation in such tissues involves differences in the basic metabolic pathways [23]. Metabolic mobilization in organogenic cultures of Adenia hondala showed a gradual increasing trend in the accumulation of proteins and starch during the initial stages of shoot formation followed by a decreasing trend during later stages of development.

Starch metabolism associated with organogenic and embryogenic process has been investigated in tissue cultures of tobacco [24], Solanum suratense [25], Papaver orientalis [26]. A positive correlation has been observed between starch accumulation and utilization during organogenesis in Adenia hondala. Starch functions as a readily available reserve for the high energy requiring process of organogenesis or as an osmotic agent in the form of free soluble sugars essentially required for the development [27].

Soluble sugar increased to higher levels during organ formation and decreased during later development stages. The increasing levels of metabolites were preceded by a short period of steady phase where the cultures underwent a conditioning process. The accumulation was followed by a gradual utilization of the reserves for organogenic requirements. In both direct and indirect organogenic cultures, the decline of metabolite accumulations started after 15 days, but in embryogenic cultures it occurred after 10 days. It is quite likely that embryo inception might take place little earlier than shoot induction. Sugars are known for their participation on many biochemical events and differentiation process. The early pool of free soluble sugar formed from sucrose hydrolysis might be a requisite in an otherwise nonphotosynthetic and energy deficient tissue for the synthesis of starch and other cellular micro- molecules requiring for initiating cell division and tissue growth [28].

In vitro techniques are now successfully applied to a range of threatened and endangered medicinal and aromatic plant species for multiplication and conservation. The present work describes an efficient multiplication and conservation system for adenia hondala using in vitro techniques

[FIGURE 1 OMITTED]

References

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[12] Hu C.Y., Wang P.J. 1983. Meristerm shoot tip and bud culture. In:Evans, D.A., Wang, W. R., Ammiratto P.V. and Yamada Y., (eds). Handbook of Plant Cell Culture, Vol. 1 Macmillan Publishing Co., New York. Pp. 177-277.

[13] Vieitez A.M., Carmen S.M. and Vieitz E. 1985. In vitro plantlet regeneration from juvenile and mature Quercus robur. L.J. Hort. Sci. 60: 99-106.

[14] Chang B. K. W. and Criley A. 1993. Clonal propagation of Pink Ginger in vitro. Hort. Sci. 28: 1203-1205.

[15] Ahuja A., Verma M. and Grewal, S. 1982. Clonal propagation of Ocimum species through tissue culture. Indian J. Exp. Biol. 20: 455-458.

[16] Fennel S., Bohorova N., Ginkel M. Van, Crosa J. and Hoisngto D.A. 1996. Plant regeneration from immature embryos of 48 elite CIMMYT bread wheats. Theor. Appl. Genetics. 92: 163-169.

[17] Anil Kumar, Sood A., Palani L.M.S. and Gupta A.K 1999. In vitro propagation of Gladiolus Hybridus Hort.: Synergistic effect of heat shock and sucrose on morphogenesis. Plant Cell Tissue Organ Cult. 57: 105 - 112.

[18] Nair L.G. and Seemi 2001 Rapid in vitro multiplication and restoration of Celastrus paniculatus Willd. Subsp. Paniculatus (Celesateracea), a medicinal woody climber Indian J. of Experimental Biology. 39: 697-704.

[19] Ajith Kumar D. and Seemi S. 1998. Rapid clonal propagation through in vitro axillary shoot proliferation of Aegle marmelos (L.) Corr., a medicinal tree. Plant Cell Rep 17: 422-426.

[20] Fernandez-Gujarro B., Celestino O., Torbio M. 1995. Influence of external factors on secondary embryogenesis and germination in somatic embryos from leaves of Qercus suber L. Plant Cell Tiss. Organ Cult 41: 99-106.

[21] Huetteman C. A., Preece and J. E 1993. Thidiazuron: a potent cytokinin for woody plant tissue culture. Plant Cell Tiss. Org. Cult. 33: 105-119.

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[23] Dey M., Kalia S., Gosh S. and Mukherjee S.G. 1998 Biochemical and molecular basis of differentiation in plant tissue culture. Current Science vol. 74: 7.

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[25] Swarnakar P.L, Bohra S.P., Chandra N. 1986 Biochemical changes during plant growth differentiation of callus of Solanum suratense. Journal of plant physiol. 126: 75-81.

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A. Aruna, Joy P. Joseph * and Ginu Joseph

Centre for Research, Dept. of Botany, Sacred Heart College, Thevara, Kochi, Kerala--682013, India

* Email: joyjoyp@rediffmail.com
Table 1: Organogenesis From Young Nodal Explants and Calli
of Adenia hondala on MS Medium Containing Different PGR Combinations.

                                             % of      Days to
SI.                                        cultures     induce
No.    Growth Regulator (mg    Explant    initiating    shoots
               I"1)                         callus

1     MS Basal                   Node         75          7
2     BAP 0.5                    Node        100          6

3     KnO.5                      Node        100          8
4     2iP 1                      Node         75          9
5     BAP 0.5 + Kn 0.5           Node        100          5

6     BAP 0.5 + Kn 0.5 +         Node        100          6
      10% CW
7     BAP 0.5 + 2iP 0.5          Node        100          6
8     BAP 0.5                   Callus        90          5
9     KnO.5                     Callus        85          6
10    BAP 1+ Kn 0.5             Callus       100          7
11    BAP 1+ Kn 0.5 +           Callus       100          7
      10% CW
12    BAP 1 +2iP 0.5            Callus       100          6

            Mean no.              Mean Length
SI.         of shoots           of shoots after
No.         after 30          45 days [+ or -] SD
        days  [+ or -] SD            (Cms)

1        1 [+ or -] 0.3         1.5 [+ or -] 0.3
2       4.25 [+ or -] 0.9      2.17 [+ or -] 0.1

3      2.75 [+ or -] 0'.5      2.52 [+ or -] 0.3
4       2.5 [+ or -] 0.5       2.25 [+ or -] 0.6
5       6.25 [+ or -] 0.5      4.75 [+ or -] 0.6

6       8.25 [+ or -] 0.9      5.15 [+ or -] 0.8

7        5 [+ or -] 0.81       8.25 [+ or -] 0.6
8        7 [+ or -] 0.4         5.1 [+ or -] 0.4
9        6 [+ or -] 0.8        4.65 [+ or -] 0.5
10     11.25 [+ or -] 0.9      6.92 [+ or -] 1.1
11     14.25 [+ or -] 1.5      11.75 [+ or -] 2.5

12      7.75 [+ or -] 0.9      7.625 [+ or -] 0.3

Table 2: Somatic embryogenesis from different explants of
Adenia hondala on MS medium containing different PGR combinations.

SI.                                         % of cultures      Days to
No.     Explant      Growth regulator     initiating callus     induce
                         (mg I"1)                              embryos

1     Leaf          NAA 2 + BAP                   85              25
                    0.5
2     Leaf          NAA 2 + BAP                   75              45
                    0.5 + CH 200
3     Internodal    NAA 1+BAP 0.5                 85              25
      callus
4     Internodal    NAA 1+ BAP                    80              28
      callus        0.5+ TDZ 0.5
-5    Internodal    2, 4-D 2 + Kn 1               75              28
      callus
6     Embryo        NAA 0.05+ BAP                 72              14
      maturation    1+6% Sucrose
7     Embryo        NAA 0.05+BAP                  36              34
      maturation    1+GA3 1.5 +6%

SI.       Mean no. of
No.         embryos
            after 30
        days [+ or -] SD

1       8.5 [+ or -] 0.5
2       34 [+ or -] 0.2
3          59.75+4.5
4      62.4 [+ or -] 2.5
-5          11.5+1.2
6           17.3+0.7
7      25.3 [+ or -] 1.8
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Author:Aruna, A.; Joseph, Joy P.; Joseph, Ginu
Publication:International Journal of Biotechnology & Biochemistry
Article Type:Report
Geographic Code:9INDI
Date:Dec 1, 2009
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