Indian Journal of Biotechnology Vol 5, October 2006, pp 527-534
Assessment of variability in the regenerants from long-term cultures of
‘safed musli’ (Chlorophytum borivilianum)
Dilip K Arora, Sarabjeet S Suri and K G Ramawat*
Laboratory of Bio-molecular Technology, Department of Botany, M L Sukhadia University, Udaipur 313 001, India Received 7 January 2005; revised 3 October 2005; accepted 5 December 2005
Somatic embryogenesis in long-term calluses of seedling and leaf explants, obtained from in vitro grown plants of Chlorophytum borivilianum—an endangered medicinal herb, has been achieved. Large number of plants were established in the field and evaluated for variability among the regenerants. The plantlets obtained through seedling-derived embryonic callus showed high level of morphological and cytological variations, which increased with the increase in age of cultures.
Occasionally, variegated plants were also observed. Variation in leaf size, stomatal number, epidermal cell size and chromosomal number was observed in regenerants. Chromosomal variation was the least (3X-3 to 3X+3) in regenerants from 1 to 4-month-old cultures and increased (5X-1 to 7X) with the age in regenerants from cultures older than 6 months.
Alternatively, regenerated plants from somatic embryos recurrently obtained from leaf explants showed very little variation (0.62%) in RAPD fingerprinting. However, further improvement in somatic embryogenesis is required for domestication of the plant using biotechnological method of propagation.
Keywords: Chlorohphytum borivilianum, RAPD, somaclonal variation, somatic embryos IPC Code: Int. Cl.8 A01H4/00; C12N15/10
Introduction
Chlorophytum borivilianum Sant. & Fern.
(Liliaceae), along with some of its other species, is commonly known as ‘safed musli’ (meaning white roots). Its tuberous roots are widely used as tonic and aphrodisiac for the presence of steroidal saponins, such as neotigogenin, neohecogenin, stigmasterol and tokorogenin1-3. The saponin fractions from dried roots showed significant inhibition of 3H-dopamine uptake in striatal syneptosomes of rats; thereby, directly stimulating the neuronal activity of dopaminergic system in the brain (unpublished).
In nature, C. borivilianum plant is propagated by seeds and tuberous roots, but it encounters the problem of low seed set, viability and germination.
Further, the population of the plant is dwindling at an alarming rate due to over-exploitation from the wild stands4,5, resulting it into an endangered species6. Therefore, the immediate task is to multiply the plant in bulk amount required for its conservation. Plant
tissue culture has been successfully used to micropropagate many medicinal plants and several other members of Liliaceae7-13. Plantlet regeneration has been reported through apical meristem and stem- disc explants in C. comosum var. folis medio variegatis14 and C. borivilianum15-17, respectively.
Purohit et al15 reported the multiplication of C.
borivilianum plants through organogenesis, which was subsequently been improved by Suri et al10. In vitro tuber formation has also been reported in the species that resulted in 100% survivability of plantlets on transfer to soil10. While attempts made by Bordia et al4 to develop field methods of multiplication and propagation through seeds and/or tuberous roots have met with failure. Somatic embryogenesis in callus cultures and decline of embryonic potential with the age of cultures was reported in C. borivilianum18. In an effort to develop technology for large-scale multiplication of C. borivilianum, conditions for shoot multiplication was optimized by using 22.2 μM BA with phytagel (0.2%), encapsulation, etc and 15,000 plantlets were produced. These plantlets showed about 90% survival in field conditions and produced tubers and were cost effective16,19,20. Members of the family Liliaceae, like onion, garlic, lilies, have been cultured in vitro and problems like variation in
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*Author for correspondence:
Tel & Fax: 91-294-2425010 E-mail: kg_ramawat@yahoo.com
Abbreviations: 2,4-D-2,4-dichlorophenoxy acetic acid; Kn- kinetin; BA-6, benzyl adenine; IAA-indole acetic acid; TDZ- thidiazuron
response of different cultivars, limitation of suitable explant, and maintenance of long-term embryonic cultures have been experienced21-22. The occurrence of cryptic genetic defects arising via somaclonal variation in the regenerants can seriously limit the utility of micropropagation protocol. Therefore, it is important to establish the suitability of a particular micropropagation protocol for a particular clone developed with respect to the production of genetically identical and stable plants before it is released for commercial purpose. No efforts have been made so far for characterization of variation in tissue culture derived plantlets of C. borivilianum.
The present work reports the screening of plants raised through long-term embryogenic cultures of C. borivilianum and development of a method for the maintenance of genetically stable stock through leaf explant culture.
Materials and Methods
Somatic Embryogenesis
Embryogenic callus was obtained from two sources: 1) root-shoot junction of seedlings and 2) stem disc-derived callus on B5 medium23, supple- mented with 2.25 μM 2,4-D and 1.15 μM Kn, as de- scribed earlier18. The cultures were maintained on the same medium by sub-culturing after 4-weeks growth.
The medium was gelled with 0.8% (w/v) bacterio- logical agar (Himedia) and pH of the medium was adjusted to 5.8 with 0.5 N HCl or NaOH and 33 mL medium was dispensed into each conical flask (100 mL, ‘Borosil’). Culture flasks were closed with non- absorbent cotton and autoclaved for 15 min at 121°C (1.05 kg cm-2)
Germination of Somatic Embryos
Somatic embryos formed from seedling- and stem disc-derived calluses were isolated under a stereo mi- croscope and transferred onto MS-9 medium [MS medium containing 400 mg N L-1 KNO3 and 100 mg N L-1 (NH4)2SO4)] containing 10 μM BA as described earlier18. In vitro germinated somatic embryos grew in clusters and maintained on the same medium. This resulted in development of clusters of shoots. Since the germination of somatic embryos required a cyto- kinin, root development was poor.
Multiplication of Propagule
Shoots obtained by the germination of somatic em- bryos were used for the multiplication and mainte- nance of cultures. A single shoot or cluster of 10
shoots was transferred onto MS-9 medium containing 25 mg L-1 adenine, 4.4 to 22.2 μM BA and 0.57 μM IAA. Leaves were harvested from 1-4-month-old in vitro plantlets and were used as a source of explant to initiate further somatic embryogenesis on MS-9 me- dium containing 12.3 μM TDZ and 1.13 μM 2,4-D.
All the experiments were conducted using 10 repli- cates per treatment, repeated twice and data were ana- lyzed statistically using ANOVA test with one or two variables, as the case may be. Cultures were incubated at 26 ± 0.5°C under white fluorescent light (Philips cool TL 36 W/54, 220 V) with a total irradiance of 36 μ mol m-2 S-1 for 16 h photoperiod and 60% relative humidity. Observations were recorded after 4-weeks of growth.
Rhizogenesis and Field Transfer of Miniature plantlets
Shoots produced in vitro by germination of somatic embryos were rooted in B5 medium supplemented with 0.57 μM IAA as described earlier10. Miniature plantlets were transferred in plastic pots (150 mL) containing sterilized garden soil-vermiculite mixture (2:1, v/v). To maintain high ambient humidity (70- 80%), pots were covered with transparent polyethyl- ene bags containing a few pores to allow gaseous ex- change. Plants were irrigated as and when required by tap water (25 mL/pot). After 15 d growth in soil, polyethylene bags were removed and plants were ex- posed to sunlight for 2 h/d for two weeks and then kept in moist and shady place. One-month-old plant- lets were finally transferred in the field and exposed to external environment.
Thus, plantlets obtained through embryogenesis from leaf and seedling calluses were available for investigation of variation.
Morphological and Cytological Variations in Plantlets
Plants obtained through somatic embryogenesis were screened for morphological and cytological variations. The plantlets from two successive lots were analyzed for variation and data are average of at least 10 plantlets. Data from all the plants were grouped into three categories (A,B,C) on the basis of leaf size (A= 2-3 cms, B= 3.5-4.5 cms, C= 6-7 cms) for comparison. Actively growing root tips of germi- nated embryos were pretreated with a saturated aque- ous solution of p-dichlorobenzene for 3 h at 25°C, washed in distilled water and fixed in a solution of acetic acid and absolute ethyl alcohol (1:3, v/v) for 24 h. Metaphase chromosomes were counted in smears as described earlier18.
ARORA et al: VARIABILITY IN REGENERANTS OF SAFED MUSALI 529
RAPD Fingerprinting
Total DNA was extracted from young leaves of micropropagated plants (3 to 5-month-old), raised through leaf-derived embryogenic calluses, and the mother plant from which the explants were prepared by modified CTAB method24. DNA was quantified by measuring absorbance at 260 nm. Ten arbitrary deca- mer primers (Operon Technologies Inc., Almeda, California) from kits A, C and K were used for ran- dom amplified polymorphic DNA (RAPD) finger- printing of the micropropagated plants, raised through embryogenic callus derived from a single leaf explant.
Each 25 μL reaction volume contained 10 mM Tris- HCl (pH 9.0), 50 mM KCl, 2.5 mM MgCl2, 0.20 mM each of dNTPs (Promega, Madison), 15 ng of the primer, 1×Taq polymerase buffer, 0.5 units of Taq DNA polymerase (Promega), and 30 ng of genomic DNA template. The reaction mixture was overlaid with an equal volume of mineral oil (Sigma). DNA amplification was performed in Perkin-Elmer Cetus 480 DNA thermal cycler (Perkin-Elmer Cetus, Nor- walk, USA). The thermocycler was programmed for one cycle of initial denaturation at 94°C for 5 min, followed by 45 cycles of denaturation at 94°C for 1 min, annealing at 34°C for 30 sec and polymerization at 72°C for 1 min. Final extension was carried out at 72°C for 10 min. Electrophoresis of amplified PCR products was performed at 60 amp for 90 min in 1.2%
(w/v) agarose (Pharmacia) gel with 1X TAE buffer, stained with ethidium bromide, and photographed under UV light. Lambda DNA digested with Hind III (Genei, India) was used as molecular size marker. The frequency of polymorphism was calculated by divid- ing the variation observed in bands to total bands re- corded through using four primers in 16 plants.
Results and Discussion
Somatic Embryogenesis and Multiplication of Propagule
Seedling- and stem disc-derived calluses produced somatic embryos on B5 medium supplemented with 2.25 μM 2,4-D and 1.15 μM Kn. These somatic em- bryos germinated to produce shoots on transfer to MS-9 medium containing 22.2 μM BAP, but roots remained undeveloped (Fig. 1a). Therefore, these shoots were allowed to grow and multiply on the same medium for 8 weeks. Further, to optimize the multiplication rate, shoots were inoculated either sin- gly or in a cluster of 10 on MS-9 medium supple- mented with different concentrations of BAP (4.4–
22.2 μM) (Table 1). Although higher concentration (22.2 μM) of BAP resulted in high rate of shoot pro- liferation, the size of inoculum also played an impor- tant role in shoot multiplication. Inoculum of a single shoot produced 3.8 shoots, whereas a cluster of 10 shoots produced 20 shoots. The effect of BA, inocu- lum size and their interactions on shoot proliferation was found statistically significant. Therefore, separa- tion of shoots obtained by the germination of somatic embryos was desirable for higher rate of multiplica- tion.
The leaf explants from in vitro-grown shoots were transferred on various media, which failed to produce callus and somatic embryogenesis (negative data not presented), except on medium containing TDZ (Table 2). It was also recorded that callus produced from the leaf explants did not survive on subculture, except those obtained from the basal region of the leaf. Ini- tially, callus grew slowly producing embryos at 4 weeks growth. However, this callus grew faster dur- ing next 6-8 weeks on the same medium and during this extended period some embryos germinated.
About 8 embryos per explant with 25% explant re- sponse was recorded on the medium with 34 μM TDZ (Fig. 1b).
Single shoot obtained by the germinating somatic embryo produced a bunch of multiple shoots on MS-9 medium containing 22.2 μM BA and leaf explants from these plantlets produced somatic embryos on TDZ (34 μM) containing medium. Thus, a continuous system of multiplication and regeneration was estab- lished.
Rhizogenesis and Field Transfer of Plantlets
Shoots produced in vitro by germination of somatic embryos were rooted on B5 medium supplemented with 0.57 μM IAA (Fig. 1c). Such plantlets were suc- cessfully transferred in soil with a high rate of surviv- ability (90%). Starting from 10 g fresh seedling- derived callus, >3000 somatic embryos could be pro- duced, of which 50% embryos germinated in vitro into plantlets and >1300 plantlets were successfully transferred in the soil (Fig. 1d). Plants developed through somatic embryogenesis from 1 to 4-month- old cultures were comparable to in vivo material in terms of vigour and yield.
Morphological Variations in Regenerants
Plantlets obtained through somatic embryogenesis were compared for their morphological characters, viz. leaf length, leaf width, size of stomata, number of
Fig. I (a-e)-a. Clumps of germinated somatic embryos; b. Callus formation and germinating somatic embryos from the leaf explants on medium with TDZ; c. Plantlets obtained from germinated somatic embryos; d. Potted plantlets; e. Broad and namow leaved shoots; & f.
Occasional variegated plant observed in the cultures.
stomata per unit area and epidermal cell size; 30 plants were randomly selected for the purpose. On the basis of variations recorded, plants were classified into three groups, Group A, B and C (Table 3). It is evident from the results that a significant variation exists in all the parameters used. Leaf length and width varied significantly. Plants in group C were 2.6 times longer than that of group A TFig. le). Number per mm2 and size of stomata also v ~ e d significantly;
the lowest in number and highest in size were ob- served in group C. Significant variations noticed in epidermal cell size and highest being in plants of
group C. Thus, the plants of group C had long narrow leaves with least number of stomata and large epi- dermal cells (Table 3). These observations clearly affirm that there existed considerable variations in plantlets obtained through somatic embryogenesis.
Occasionally variegated plants were also observed in cultures from long-term cultures obtained from stem disc through organogenesis (Fig. If).
Cytological Variations in Regenerants
C. borivilianum is a diploid species2hnd the chromosome number reported in it is 2n=16 with a basic number of X=8. Interestingly, a series of poly-
ARORA et al: VARIABILITY IN REGENERANTS OF SAFED MUSALI 531 ploidy in the natural population has been reported in
the species. The polyploidy within the species could be related to the predominant vegetative propagation in the species. In the present study, authors investi- gated the cytological variation in the plants regener- ated through somatic embryogenesis. Such plants maintained for long periods showed instability and
decline. The cytological investigations of root tips of germinated somatic embryos showed a wide variation in chromosome number (Table 4, Figs 2a-c). A clear tendency of increase in ploidy level during the in vitro growth of cultures is evident from the results. Varia- tion (3X-3 to 3X+3) was least in 1 to 4-month-old cultures. While somatic embryos produced in cultures older than 6 months failed to germinate, which may be due to high instability in the chromosome number and increase in ploidy level. These precociously ger- minated somatic embryos showed a wide variation in the ploidy level, ranging from 5X-1 to 7X. However, all the plants raised through organogenesis from stem disc explants, in an earlier study10, showed a stable ploidy level (3X).
Fig. 2(a-c)—a. Root tip squash of precociously germinated abnormal somatic embryo showing 56 chromosomes; b & c. Root tip squash showing polyploidy (6X & higher) in long-term cultures.
Table 1—Effect of different concentrations of BAP and explant inoculum size on shoot proliferation on MS-9 medium containing IAA (0.57 μM) and adenine (25 mg L-1)
BAP
(μM) No. of shoots per inoculum
No. of shoots proliferated±
S.D*
Shoot length cm± S.D
4.4 1 1.8 ± 0.7 7.0 ± 1.7
11.1 1 2.6 ± 0.5 6.8 ± 1.6
22.2 1 3.8 ± 0.8 7.4 ± 1.7
4.4 10 14.8 ± 0.8 5.6 ± 1.4
11.1 10 16.6 ± 1.1 5.6 ± 1.9
22.2 10 21.4 ± 1.1 6.3 ± 1.7
*Inoculum: Significant at 1%, SE = 0.48, CD1%= 1.530 BAP: Significant at 1%, SE = 0.59, CD1%= 1.91
Inoculum X BAP: Significant at 1%, SE = 0.83, CD1%= 2.6 Table 2—Effect of TDZ on somatic embryo formation from leaf explants
Medium
TDZ μM %
response
Callusing Somatic embryos/
explant
11.25 Nil Nil -
22.7 10 Cut ends -
34 25 Callus 7
Table 3—Morphological variation in plantlets generated through somatic embryogenesis from seedling-derived callus
Leaf Stomata Epidermis cell size
Group
Lengtha
cm ± S.D Widthb
cm ± S.D Numberc
mm2± S.D Lengthd
cm ± S.D Widthe
cm ± S.D Lengthf
cm ± S.D Widthg cm ± S.D A 2.50 ± 0.3 0.65 ± 0.1 11.2 ± 0.5 24.8 ± 2.2 21.5 ± 0.8 106.8 ± 39.5 36.2 ± 4.5 B 3.96 ± 0.1 0.68 ± 0.1 8.9 ± 0.5 25.1 ± 1.7 22.5 ± 1.9 107.6 ± 11.7 29.1 ± 3.3 C 6.50 ± 0.3 0.47 ± 0.1 5.3 ± 0.7 39.0 ± 3.9 25.0 ± 1.4 274.7 ± 4.8 33.7 ± 1.9
aSignificant at 1%, SE = 0.07, CD1%= 0.19
bSignificant at 1%, SE = 0.01, CD1%= 0.03
cSignificant at 1%, SE = 0.17, CD1%= 0.47
dSignificant at 1%, SE = 0.82, CD1%= 2.26
eSignificant at 1%, SE = 0.42, CD1%= 1.17
fSignificant at 1%, SE = 7.01, CD1%= 19.38
gSignificant at 1%, SE = 1.01, CD1%= 2.80
Table 4—Chromosomal variation in in vitro regenerated plantlets raised through organogenesis and somatic embryogenesis (SE) Roots Age of cultures
(months)
Number of chromosome
Ploidy level
In vitro plantlets* - 24 3X
SE 1 to 4 21 to 27 3X-3 to
3X+3 Precociously
germinated SE
18 to 24 39 to 56 5X-1 to 7X
*Plantlets raised through organogenesis from stem disc explants (Suri et al 10).
RAPD Fingerprinting
Since a high level of morphological and cytological variation was observed in the plants obtained through embryogenic calluses from seedling and stem disc explants, they were not subjected to RAPD finger- printing. The plants raised through leaf-derived em- bryogenic callus were screened for their genetic sta- bility by RAPD fingerprinting. Of the primers initially screened, OPA20 (GTTGCGATCC), OPC2 (GTGAGGCGTC), OPC13 (AAGCCTCGTC) and OPK6 (CACCTTTCCC) were short-listed for the study and each produced 5, 4, 6 and 4 scorable bands, respectively. An average of 4.7 bands was scored per primer. Sixteen regenerated plants were compared with the control mother plant. The amplified products ranged in size from 0.56 to 2.0 kb. The total number of bands scored was 323 and most of them were found identical to the mother plant, except two novel bands (approximately 0.6 and 0.75 kb) produced in two regenerants using primer OPA20. Similarly, no variation was observed with the other primers used for the study (Figs 3 a-d). The frequency of polymor- phism observed in the plants raised through leaf- derived embryogenic calluses was quite low (0.62%).
Thus, RAPD fingerprinting revealed that the tiny sec- tions of the plant genome, which were amplified by arbitrary primers, did no vary in plants obtained through somatic embryogenesis from single leaf ex- plant and these plants may be genetically uniform.
Thus, the method adopted to micropropagate C. borivilianum through somatic embryogenesis from 1 to 4-month-old cultures could be used to maintain genetic stability and to conserve true-to-type charac- ters.
Like most members of the family Liliaceae, C. borivilianum has poor seed set and seed viability, whereas propagules are required on large-scale for the domestication of the plant. Present report describes
Fig. 3(a-d)—a. Gel electrophoresis of RAPD fragments obtained in the mother plant (lane 2) and micropropagated plants of C.
borivilianum raised through somatic embryogenesis from leaf explant derived callus (remaining lanes) with the primer OPA20 (GTTGCGATCC) (Note the polymorphic fragments in lanes 4 and 12); b. Gel electrophoresis of RAPD fragments obtained with the primer OPC13 (AAGCCTCGTC) showing no variation in the regenerants and the mother plant; c. Gel electrophoresis of RAPD fragments obtained with the primer OPC02 (GTGAGGCGTC), no variation was observed; & d. Gel electrophoresis of RAPD fragments obtained with the primer OPK6 (CACCTTTCCC) showing no variation in the plantlets.
the fate of a Liliaceae plant in long-term cultures.
High variation was recorded in long-term cultures of C. borivilianum, which increased with the age of the cultures. However, a short cut method of renewed embryogenesis using leaf explants may turn useful for obtaining genetically uniform material.
Maintenance of genetic integrity in micropropaga- tion system with regard to explant source is of para- mount importance. Therefore, axillary branching or somatic embryogenesis is preferred27. Shenoy and Vasil28 revealed complete genetic stability in somatic embryo-derived plants of Pennisetum purpureum.
Shoyama et al29 reported that somatic embryogenesis-
ARORA et al: VARIABILITY IN REGENERANTS OF SAFED MUSALI 533 derived regenerants of Panax ginseng contained the
same chromosome number as the mother plant and complete stability was established by RAPD finger- printing30. Short-term cultures of C. borivilianum raised from leaf explants showed stability, while long- term cultures showed instability irrespective of origin in terms of chromosome numbers and RAPD finger- printing as observed in garlic21. This instability might be due to continuous growth of cultures in the pres- ence of 2,4-D and interaction of genome with the me- dium. The complex heterozygous nature of most of the Liliaceae plants may be the cause of this instabil- ity. In the present work, instability and decline in em- bryogenic potential was a major problem with the seedling-derived callus for long-term maintenance.
This problem was, however, overcome by the use of callus obtained from the leaf explants that forms a cyclic system of shoots → callus → embryogenesis
→ germinated embryos → leaf → callus.
Acknowledgement
Authors wish to thank Prof S N Raina, Department of Botany, Delhi University, Delhi for RAPD analysis and funds received under ICAR (ad-hoc scheme), DST-FIST and UGC-DRS programmes to KGR.
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