a critically endangered medicinal plant of t

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genesis have also been reported in Lithospermum erythrorhizon of the same family (Yu et al., 1997). Root formation. Harvested regenerated shoots were placed ...
In Vitro Cell. Dev. Biol.—Plant 41:244–248, May–June 2005 q 2005 Society for In Vitro Biology 1054-5476/05 $18.00+0.00

DOI: 10.1079/IVP2004612

ORGANOGENESIS, EMBRYOGENESIS, AND SYNTHETIC SEED PRODUCTION IN ARNEBIA EUCHROMA – A CRITICALLY ENDANGERED MEDICINAL PLANT OF THE HIMALAYA SUMIT MANJKHOLA, UPPEANDRA DHAR*,

AND

MEENA JOSHI

G. B. Pant Institute of Himalayan Environment and Development, Kosi-Katarmal, Almora-263 643, Uttaranchal, India (Received 14 May 2004; accepted 20 October 2004; editor A. Pretova´)

Summary This is the first report of simultaneous organogenesis and somatic embryogenesis in Arnebia euchroma, a highly valued, critically endangered medicinal plant of the Himalaya. Root-derived callus showed only rhizogenesis, whereas leafderived callus showed simultaneous organogenesis and somatic embryogenesis. Organogenesis was optimal (12.2 shoots per culture) in 1 mM indole-3-butyric acid combined with 2.5 mM 6-benzyladenine and induction of somatic embryogenesis (16.3 embryos per culture) occurred in 2.5 mM indole-3-butyric acid combined with 2.5 mM 6-benzyladenine. Shoots rooted (100%) best in half-strength Murashige and Skoog (MS) medium supplemented with 2.0 mM indole-3-butyric acid. Early cotyledonary-stage embryos encapsulated with 3% sodium alginate and calcium nitrate (100 mM for 25 min) showed 60.6% germination in MS medium. Rooted shoots transferred to a mixture of sterile soil, sand, and peat (1:1:1 by volume) showed 72% survival ex vitro. Application of these protocols would be helpful in reducing pressure in natural populations, in genetic transformation studies, and in long-term storage of elite genotypes through synthetic seed production. Key words: Arnebia; organogenesis; secondary embryogenesis; somatic embryos. Considering the above, in vitro plant regeneration could help in the conservation and sustainable use of the species. It might help in meeting the requirements of shikonin production. A. euchroma tissue culture was pioneered in Russia (Davydenkov et al., 1991). Until recently, tissue culture of A. euchroma has largely focused on improving shikonin production (Sokha, 1996; Zakhlenjuk and Kunakh, 1998). In addition to conservation, the establishment of a reliable in vitro regeneration protocol for A. euchroma is essential for the recovery of transgenic plants and for application of genetic engineering techniques to plants, for example, to enhance pigment production (Yu et al., 1997). The purpose of the present study is to (1) develop an efficient plant regeneration system in A. euchroma via organogenesis and somatic embryogenesis and (2) explore possibilities of developing synthetic seed production systems and subsequent germination protocols of synthetic seeds.

Introduction Arnebia euchroma (Royle) Jonst. (Boraginaceae) is a perennial plant of the alpine region distributed in the Pamirs, the Tien Shan, the Himalaya and western Tibet between an altitudinal range of 3700 and 4200 m above sea level (asl; Anonymous, 1985). Shikonin and its derivatives extracted from the roots of A. euchroma have been known since ancient times and used as dyes for silk and food products. Shikonin, valued at US$4000 kg21 (wholesale price) possesses antibacterial, antifungal, anti-inflammatory, and woundhealing properties. The species is also used in various diseases of the tongue and throat as well as fevers and cardiac disorders (Terada et al., 1990). Furthermore, A. euchroma exhibits potent anti-HIV activity (Kashiwada et al., 1995). Arnebin 1 and arnebin 3 derived from it possess anticancerous properties (Harborne and Baxter, 1996). Because of its medicinal uses, the species is being harvested indiscriminately from the wild both for domestic and pharmaceutical purposes. This has resulted in A. euchroma’s critically endangered status and its listing in the species prioritized for conservation in West Himalaya (Molur and Walker, 1998). Roots of the species are being harvested from natural populations without considering the reproductive status of the individual plants. A large number of sexually immature plants are collected, thereby affecting the seed set. A. euchroma is reported to possess level III difficulty (very difficult) in propagation (Molur and Walker, 1998) and so far, it is not being cultivated anywhere in the Himalayan region.

Materials and Methods Plant material and sterilization. Seeds of A. euchroma were collected at maturity from natural populations of Malari (3615 m asl), Uttaranchal, West Himalaya. Seeds were dried at room temperature and stored at 48C until use for experiments. Seed coats were removed and seeds were washed with Tween 20 (Hi-Media, Bombay) for 10 min followed by washing in tap water, then rinsed five times with double-distilled water. Seeds were surfacedisinfected for 9 min in 0.1% HgCl2 followed by thorough rinsing with sterile double-distilled water. For in vitro germination, the seeds were inoculated in MS basal medium (Murashige and Skoog, 1962) with 3% sucrose and 0.7% bacteriological agar as a gelling agent. Media preparation and culture conditions. All media were adjusted to pH 5.7 prior to autoclaving at 1218C for 22 min. All the chemicals used were of analytical grade (Sigma Chemical Co., St. Louis, MO, USA;

*Author to whom correspondence should be addressed: Email udhar@ nde.vsnl.net.in

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ORGANOGENESIS AND EMBRYOGENESIS OF A. EUCHROMA Merck and Hi Media, Mumbai, India). The culture tubes (25 £ 150 mm; Borosil, India) containing 15 ml of medium and conical flasks (100 ml; Borosil, India) with 40 ml of medium were plugged with non-absorbent cotton plugs. All cultures were maintained at 25 ^ 28C in a 16 h light and 8 h dark photoperiod, with irradiance (40 mmol m22 s21) by cool fluorescent tubes. Organogenesis and embryogenesis. For callus induction, leaf and root explants from aseptically raised seedlings (1 mo.) were used. Leaf explants (0.5 £ 0.5 cm) were placed abaxially, and root explants (about 1 cm) were placed horizontally in the medium. The callus induction medium was comprised of MS basal medium (Murashige and Skoog, 1962) containing 3% sucrose, 0.7% agar, and 0.1 g l21 casein hydrolyzate supplemented with different concentrations of 2,4-dichlorophenoxy acetic acid (2,4-D; 0, 1, 2.5 mM) and indole-3-butyric acid (IBA; 0, 1, 2.5 mM) in combination with different concentrations of 6-benzyladenine (BA; 0, 0.5, 1.0, 2.5, 5.0 mM) (Table 1). These treatments were based on the promising results obtained during experimental trials and on the basis of published reports on related species. After 6 wk of inoculation, the frequency of callus formation was recorded. The callus (approximately 400 mg) obtained from different combinations was transferred at 4-wk intervals for 2 mo. until regeneration occurred. Data on number of shoots and number of somatic embryos per culture was recorded at 4-wk intervals of the second subculture. The regenerants with well-developed leaves were recorded as shoots. The bipolar structures were scored as somatic embryos, and the total number of regenerants was estimated by adding the number of shoots and somatic embryos. Rooting. For root induction, the shoots (four or five leaves) were harvested and inoculated on full or half-strength MS medium containing 3% sucrose and 0.7% agar supplemented with different concentrations of IBA (0, 1.0, 1.5, 2.0, 2.5 mM). Culture shoots were maintained under similar conditions as described previously in the Materials and Methods. Data on percentage rooting, root number, and root length were recorded after 4 wk of inoculation in rooting medium. Encapsulation and germination. Early cotyledonary-stage somatic embryos (5 –6 mm long) obtained were encapsulated in sodium alginate (3%) and calcium nitrate (100 mM for 25 min). The alginate beads containing somatic embryos were washed three times with sterilized distilled water and wiped dry with filter paper. Encapsulated and non-encapsulated somatic embryos were inoculated in full or half-strength MS medium with 3% sucrose and 0.7% agar, supplemented with IBA (0, 2.5 mM) in combination with BA (0, 2.5 mM). Data on the percentage of germination and secondary embryogenesis were recorded after 6 wk of inoculation.

TABLE 1 CALLUS INDUCTION IN ARNEBIA EUCHROMA ON DIFFERENT COMBINATIONS OF PLANT GROWTH REGULATORS (DATA RECORDED AFTER 6 wk OF INOCULATION)

Treatment code T0 T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15 T16 LSD (P , 0.05)

2,4-D (mM)

IBA (mM)

BA (mM)

0 1 1 1 1 2.5 2.5 2.5 2.5 – – – – – – – –

0 – – – – – – – – 1 1 1 1 2.5 2.5 2.5 2.5

0 0.5 1.0 2.5 5.0 0.5 1.0 2.5 5.0 0.5 1.0 2.5 5.0 0.5 1.0 2.5 5.0

Leaf (% callus induction)

Root (% callus induction)

0 26.2 39.4 46.6 53.3 67.1 90.7 86.0 80.6 46.6 66.6 67.1 73.8 90.7 97.2 100.0 100.0 10.7

0 20.0 26.2 38.0 60.0 73.0 97.6 90.7 86.1 32.9 53.3 73.8 80.0 97.6 100.0 97.6 86.0 9.8

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Acclimatization. Well-rooted shoots of A. euchroma (derived from organogenesis) were removed from culture tubes after 4 wk in rooting medium. Fifty rooted shoots were washed thoroughly with tap water to remove the adhering medium and transferred to plastic pots containing a mixture of 150 g (w/v) of autoclaved soil, sand, and peat (1:1:1 by volume). Potted plantlets were initially kept in a growth chamber. Plantlets were covered with polythene bags (for up to 45 d) to maintain high humidity and irrigated with quarter-strength MS basal salt solution (up to 1 wk), followed by tap water. After 45 d, and upon commencement of new leaves, the covers were removed permanently and hardened plantlets were transferred to nursery conditions. Statistical analysis. All the experiments were set up in a randomized complete block design. For each treatment, five explants with three replicates each were used, and each experiment was repeated at least once. Data scored in percentages were subjected to arcsine transformation before analysis and then converted back to percentages for presentation in the tables and figures. The data were subjected to ANOVA (Wilkinson, 1986) and Fisher’s least significance difference (LSD) (P , 0.05) to determine whether treatment effects were significant (Snedecor and Cochran, 1968).

Results and Discussion Callus induction. In both leaf explants as well as root explants, callus was induced in all the tested combinations of plant growth regulators (PGRs). However, time taken for callus induction, frequency of callus induction, and color and texture of callus formation varied between the explant types and different combinations of PGRs. No callus formation occurred in PGR-free medium. The frequency of callus formation in different explant types for different combination of PGRs is given in Table 1. In root explants, callus induction occurred after 16 d of inoculation, whereas in leaf explants, callus was induced after 20 d. A 100% rate of callusing was obtained on T15 (2.5 mM IBA þ 2.5 mM BA) and T16 (2.5 mM IBA þ 5.0 mM BA) medium in leaves and on T14 (2.5 mM IBA þ 1.0 mM BA) medium in roots. Though the frequency of callus induction was the same in T15 and T16, the callus mass was higher in T15. Overall, the frequency of callus induction was more significantly (P , 0.05) affected by the explant type than by most of the treatments used (Table 1). Creamish compact callus was formed from root explants and light yellow friable callus was formed from leaf explants. Such types of variation in response to different explant type has been reported earlier (Luo and Jia, 1998; Rout et al., 1999). Regeneration. The callus had to be subcultured twice at 4-wk intervals before it showed signs of regeneration. Leaf-induced yellow friable callus turned green and friable, whereas callus obtained from root explants remained creamish and compact throughout the cultivation. No regeneration occurred in root-derived callus. Root-derived callus showed a reddish tinge on T12 (1.0 mM IBA þ 5.0 mM BA) and rhizogenesis on T13 (2.5 mM IBA þ 0.5 mM BA). In spite of repetitive subculturing for more than 6 mo., plantlet regeneration did not occur in root-derived callus. Root-derived callus had a reddish tinge, which darkened in color. Such observations have been reported earlier for A. euchroma callus lines using dormant buds as explant sources. It is reported that calluses with a reddish tinge have higher levels of shikonin content (Zakhlenjuk and Kunakh, 1998). The growth hormone combination 2,4-D– BA did not induce differentiation, which has also been reported for other species (L. erythrorhizon) of the same family (Boraginaceae) where 2,4-D was not effective in inducing plant regeneration (Yu et al., 1997). In leaf-derived callus, simultaneous organogenesis and somatic embryogenesis was observed on different combinations of IBA and BA (Table 2).

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MANJKHOLA ET AL. TABLE 2

EFFECT OF DIFFERENT GROWTH REGULATOR COMBINATIONS ON SOMATIC EMBRYOGENESIS AND SHOOT ORGANOGENESIS IN ARNEBIA EUCHROMA (DATA RECORDED AFTER 8 wk OF CALLUS SUBCULTURE) Treatment code T0 T9 T10 T11 T12 T13 T14 T15 T16 LSD P , 0.05

Somatic embryos per culture 0f 0f 2.7 3.4 6.7 10.7 14.1 16.3 8.7 2.1

ef ef cde bc ab a cd

Shoot number per culture

Total number of regenerants

0g 6.2 cd 7.8 c 12.2 a 11.3 a 5.7 cd 4.3 de 3.5 ef 2.7 ef 14.6

0 6.2 10.5 15.6 18.0 16.4 18.4 19.8 11.4

Data followed by different letters within columns differ significantly (P , 0.05).

FIG . 1. A, Effect of different IBA concentrations on the percentage of shoots forming roots on MS and half-strength MS (1/2 MS) medium (LSD: 13.2). Bars represented by different letters differ significantly at P , 0.05 (data recorded after 4 wk of inoculation in rooting medium). B, Effect of different IBA concentrations on root number on MS and 1/2 MS medium (LSD: 2.3). Bars represented by different letters within each medium differ significantly at P , 0.05 (data recorded after 4 wk of inoculation in rooting medium).

The highest regeneration efficiency (19.8 regenerants per culture) was obtained on MS medium supplemented with 2.5 mM IBA and 2.5 mM BA. In this combination, the frequency of embryogenesis was predominant compared to organogenesis. The highest number of somatic embryos per culture (16.3) was obtained on 2.5 mM IBA and 2.5 mM BA, which was a significant (P , 0.05) improvement compared to the control and most of the treatments used except T14 (2.5 mM IBA and 1.0 mM BA). Globular embryos developed on the callus surface, which developed into early green cotyledonary leaves. The development of globular to cotyledonary-stage embryos was asynchronous. At times, cup-shaped fused cotyledons were also formed. It is reported that blocking in auxin polar transport leads to the fused cotyledon phenotype (Liu et al., 1993). The highest number of shoots per culture (12.2) via organogenesis was obtained with the combination of 1.0 mM IBA and 2.5 mM BA, which showed significant (P , 0.05) improvement over other combinations used except T12 (1.0 mM IBA þ 5.0 mM BA). No regeneration occurred in the control. Simultaneous organogenesis and somatic embryogenesis have also been reported in Lithospermum erythrorhizon of the same family (Yu et al., 1997). Root formation. Harvested regenerated shoots were placed on rooting medium consisting of MS and half-strength MS basal medium with different combinations of IBA. They showed 100% rooting on half-strength MS basal medium (Fig. 1A) with 2.0 mM

FIG . 2. A, Percentage germination in encapsulated and non-encapsulated somatic embryos (H: 2.5 mM IBA þ 2.5 mM BA) (LSD: 14.8). Bars represented by different letters differ significantly at P , 0.05 (data recorded after 6 wk of inoculation). B, Secondary somatic embryogenesis in encapsulated and non-encapsulated somatic embryos (H: 2.5 mM IBA þ 2.5 mM BA) (LSD: 12.5). Bars represented by different letters differ significantly at P , 0.05 (data recorded after 6 wk of inoculation).

ORGANOGENESIS AND EMBRYOGENESIS OF A. EUCHROMA

FIG . 3. Stages in Arnebia euchroma cultures in vitro: (A) callus formation; (B) embryogenic callus; (C) globular embryos (marked with arrow); (D) different stages of somatic embryos; (E) synthetic seed germination; (F) shoots via organogenesis; (G) rooted plantlet.

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and 2.5 mM IBA. This was a significant (P , 0.05) improvement over the control and most of the other concentrations of IBA used. On MS medium with 2.0 mM IBA, 97.1% rooting was observed. With respect to root number per explant, the highest number of roots was formed on 2.0 mM IBA, which was significantly (P , 0.05) better than most of the other concentrations used (Fig. 1B). Thus, half-strength MS basal medium with 2.0 mM IBA responded best with respect to both rooting percentage (100%) as well as root numbers (9.6 per explant). A favorable response of halfstrength MS basal medium supplemented with IBA for rooting has been reported for other herbaceous species as well. With IBA alone, rooting was induced in A. benthamii (Manjkhola, 2002). Synthetic seed. An important application of somatic embryos is their use in the production of synthetic seeds (Merkle et al., 1990). Encapsulation of embryos is the first major step in synthetic seed production. In A. euchroma, sodium alginate (3%) and calcium nitrate (100 mM for 20 min) were effective for encapsulation of synthetic seeds. Low concentrations (1– 2%) of sodium alginate beads were too soft to handle, and at higher concentration (4%) the beads were too hard and hindered the emergence of roots and shoots. When encapsulated and non-encapsulated somatic embryos were kept for germination, the maximum germination percentage of encapsulated (60.6%) and of non-encapsulated somatic embryos (73.8%) occurred on hormone-free MS medium. However, the germination percentage was not significantly different from that of half-strength MS hormone-free medium (Fig. 2A). In MS medium as well as in half-strength MS medium supplemented with 2.5 mM IBA and 2.5 mM BA, the encapsulated embryos showed a significantly (P , 0.05) lower germination percentage. In this combination, secondary embryogenesis was observed on both MS medium as well as half-strength MS medium (Fig. 2B). Lower germination percentages of encapsulated somatic embryos in comparison to non-encapsulated somatic embryos is in conformity with the findings of several researchers (Bapat and Rao, 1988; Ghosh and Sen, 1994). The calluses maintained their embryogenic capacity for more than 1 yr. Non-encapsulated somatic embryos showed faster growth than encapsulated somatic embryos. Root emergence in encapsulated somatic embryos occurred after 2 wk of inoculation and after 1 wk in non-encapsulated somatic embryos. The slower growth of encapsulated embryos can be attributed to internal diffusional limitations, which have been demonstrated for many immobilized biocatalysts (Klein et al., 1984). Acclimatization. Out of 50 plantlets transferred into pots containing autoclaved soil, sand, and peat (1:1:1 by volume), 72% survived under nursery conditions. The plants grew well and did not show any apparent morphological abnormality during the observation period of 6 mo. This is the first report on simultaneous organogenesis and embryogenesis induction in A. euchroma callus cultures and the development of synthetic seed for A. euchroma (Fig. 3). Application of these protocols would be helpful in promoting conservation of natural populations. Plant regeneration via somatic embryogenesis is advantageous for genetic transformation to obtain higher rates of production of naturally occurring pigments in A. euchroma. Additionally, synthetic seeds can be useful for storing valuable germplasm.

Acknowledgments S.M. wishes to thank Drs. R. S. Rawal, Subodh Airi, Indra Dutt Bhatt, and all his fellow colleagues of CBD Lab for their help and support. The Department of Biotechnology and Council of Scientific and Industrial Research, Government of India, are thanked for financial support.

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