Indian Journal of Experimental Biology Vol. 37, January 1999. pp. 75-82

Factors affecting sornai' ~' embryogenesis in long term callus cultures of 'safed musli' (ChlorGphytum borivilianum), an endangered wonder herb

Di lip K. Arora, Sarabjeet S. Sun , Kishan G. Ramawat & Jean-M Merillon '

Laboratory of Bio-Molecular Technology, Department of Botany, M L Sukhadia University Udaipur 31 3 00 I, India (Fax +91 294 413150)

, Plant Biotechnology Laboratory, Uni versity of Bordeaux-lI , Bordeaux, France

Received 15 April 1998; revised 6 October 1998

ChiorophYlUlIl boriniianu7Il , commonly known as ' safed musli' , has become an endangered species due to ,ts ,,:cr-exploitation for tuberous roots, used as tonic and aphrodisiac. An improved method has been ut ,'cloped for large-scale rapid multiplication of C. borivilianum through somatic embryogenesis in callus cu ltures. Somatic embryos were obtained on MS medium containing 2.25 J..lM 2,4-D and 1.15 ~lM kinetin . The concentration and com ination of KNO) and (NH4)2S04 in the medium had a marked effect on the somatic embryogenesis . Anunonium to nitrate nitrogen in the ratio of 1:4 at low total ni­trogen level (250 or 500 mgNL" ) in the medium was favourable for the growth and somatic embryo­genesis. Among the various cytokinins tested, 2-iP had a maximum stimulatory effect on somatic em­bryogenesis. Decreasing order of the effectiveness of various cytokinins incorporated in MS-9 medium (i .e., MS medium containing 100 mgNL'1 (NH4)2S0 4 and 400 mgNL'1 KJ~03) supplemented with 2.25 J..lM 2,4-0 was 2-iP > TDZ >kinetin >BAP. Incorporation of amino acids, viz. glutamine and proline, promotes embryogenesis. Maximum number of somatic embryos (63 per inoculum) were observed on MS-9 medium supplemented with 100 mgL"' proline, 1.12 J..lM 2,4-D and 7.42 J.1M 2-iP. Germination of somatic embryos, isolated from young cultures (1-5 months-old) was maximum (48%) on MS-9 me­d ium supplemented with 17.6 J.1M BAP. On the other hand, somatic embryos developed in long-term cultures were abnormal and showed a wide range of variation in the number of chromosomes, ranging from 23 to 58 (i.e" 3X-3 to 7X+2) along with several structural chromosomal abnormalities. This may be the reason for the decline in the germination potential of somatic embryos in long-term cultures. Thus, callus produced from shoot cultures obtained from stem-disc explants on B5 medium containing 2 J..lM TDZ and 0 .18 mM adenine, provides an excellent source for obtaining fresh cytologically-stable embryogenic cultures. Plamlets developed in vitro through embryo germination were successfully trans­ferred to soil with 90% survivability. The technique developed is highly efficient to get miniature plantlets for field transfer in 2 months, starting from the callus.·

Chlorophytum borivilianum (liliaceae) and a few other species are commonly known as 'safed musli'. Tuberous roots of the plant are widely used as tonic and aphrodisiac due to the presence of steroidal saponins, viz. neotigogenin, neoheco­genin, stigmasterol and tokorogenin I. 2. C. borivilianum has been used along with other plants such as Asparagus ascendense, A. racemosum, Curculigo orchioides and Withania somnifera in several formulations in the Indian system of medi­cine3. 4

• Pharmocological investigations of saponin fractions prepared from dried roots showed signifi­cant inhibition of 3H-dopamine up-take in rat striatal syncptosomes thereby, directly stimulating

the ne;,rronal activity of dopaminergic system in the brainS. Dried roots of the plant are exported from India in substantial amount and the present demand is met from forests. Population of the plant is dwindling at an alarming rate. In nature, the plant is propagated by seeds and has become an endangered species6 due to low seed set, viabii­ity and germination, associated with over­exploitation from the wild strands7

. The immediate task is to conserve and multiply the plant in bulk amount required for its domestication to meet the present demandS.

Plant tissue culture has been successfully used to micropropagate medicinal plants and several

76 rNDIAN J. EXP. BIOL. , JAl';LJARY 1999

other members of li liaceae3• 9. 10. Plantlet regenera­

tion has been reported through apical meristem in C. comosum var. Jolis medio variagatis ll and through stem-disc explant in C. borivilianumi l 2

• 13 .

Earlier, we reported induction of somatic embryos in C. borivilianum through seedling-derived cal­lus l4 . Somatic embryogenesis is a versatile tech­nique for rapid multiplication of plants and offers a superior possibility for developing scale-up tech­nology as compared to organogenesis1s. The sys­tem can be used to produce artificial seeds l6 using a bio-reactor. The present work describes a method for rapid multiplication of C. boriviliamum through high frequency somatic embryogenesis in callus cultures.

Materials and Methods Callus and somatic embryos induction Jrom

seedlings-Embryogenic callus was obtained from seedling explants on Bs medium l7 supplemented with 2.25 ~ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.15 ~ kInetin, as described earlierl4 . Cultures were maintained on the same medium by subculturing after 4-wee~s growth.

Improvement in growth and embryogenesis­(a) Effect of inorganic nitrogen source: Seedling­derived callus (ca. 200 mg fresh weight/inoculum) was transferred onto Murashige and Skoog's (MS) medium l8 containing 2.25 ~ 2,4-0, 1.l5 ~ ki­netin and different concentrations and combina­tions of KN03 (100-500 mgNL' I) and (NH.)2S0. (50-300 mgNL·1).

(b) Effect of plant growth regulators - Embry­onic cultures obtained on the above medium were transferred onto MS-9 medium (i.e. MS medium containing 400 mgNL'1 KN03 and 100 mgNL'1 (NH4)2S04) supplemented with different concen­trations and combinations of 2,4-0 (1.12-4.5 ~ and various cytokinins, viz. 2-isopentyl adenine (2-iP), 6-benzylaminopurine (BAP), thidiazuron (TDZ) or kinetin (1.45-7.42 ~.

(c) Effect of amino acids--For the rapid multi­plication and improvement of texture of somatic embryos, cultures were transferred onto MS-9 me­dium containing 1.l2 ~ 2.4-D, 7.42 ~ 2-iP and varied concentrations (10-400 mgL' I) of glutamine or proline.

Somatic embryogenesis Jrom stem-disc derived organogenetic callus--Callus was also obtained

from stem-disc derived shoot cultures of C. borivilianum maintained on Bs medium sup­plemented with 22 ~ (TOZ) and 0.18 mM ade­nine, as described earlier13

• Callus thus obtained was transferred onto MS-9 medium supplemented with 1.12 ~ 2.4-0, 7.42 ~ 2-iP and 100 mgL'1 proline. After 3 weeks growth, cultures were trans­ferred onto the germination medium.

The medium was gelled with 0.8% (w/v) bacte­riological agar (BOH). The pH of the medium was adjusted to 5.8 with 0.5 N HCI or NaOH after in·, corporation of all ingredients of the medium 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 kgCm'2).

Germination oj somatic embryos and transJer to

soil--Somatic embryos formed from seedling and stem-disc derived callus were isolated under a ste­reo microscope and transferred onto MS-9 medium devoid of plant growth regulators or containing activated charcoal (0.04-1 %), or BAP (13 .2-30.8 ~. In vitro germinated somatic embryos, main­tained for 6 weeks on MS-9 medium supplemented with 17.6 ~ BAP were washed with a solution of systemic fungicide, carbendazim (2-(methoxy car­bamoyl)-benzimidazole; 1 %, w/v) for 5 min and planted in plastic pots (150 ml) containing steril­ized garden soil : composed (1:1, '11'1) mixture. To maintain high ambient humidity (70-80%) pots were covered with transparent polythene bags containing a few pores to allow gaseous exchange. Plants were irrigated as and when required (25 mllpot, tap water). After 15 days growth in soil, polythene bags were removed and plants were ex­posed to sunlight for 2 hrlday and for rest of the period, plants were kept in moist and shady place. ' One-month-old plantlets were fina lly transferred in the field, exposed to external environment.

Cy tology--Actively growing root tips of normal somatic embryos in 1-4 months old cultures and precociously germinated abnormal embryos in 6-8 and 18-24 months-old cultures were fixed in a saturated aqueous solution of p-dichlorobenzene for 3 hr at 25°C, washed in distilled water and fixed in a mixture of acetic acid: absolute ethyl alcohol (1 :3 , v/v) for 24 hr. Root tips were hydro­lyzed with IN HCI at 60°C for 10 min, washed in distilled water, stained with 0.5% basic fuschin for

ARORA et a/.: SOMA TIC EMBRYOGENESIS IN CHLOROPHYTUM 77

45 min. and squashed in 1 % acetocannine. Num­ber of chromosomes at metaphase were counted under a light microscope (1000X magnification).

Culture conditions-All the experiments were conducted using 10 replicates in each treatment. 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 )lmolm-2S- ' for 16 hr photo-period and observations were recorded after 4-week growth.

Results and Discussion An improved method for high-frequency micro­

propagation using somatic embryogenesis in callus cultures of C. borivilianum is developed as com­pared to micropropagation through organ ogene­sis '2. 13 . Superiority of somatic embryogenesis over organogenesis for large-scale multiplication of a species is well established IS. High rate of multipli­cation along with better survivability of in vitro raised plantlets in soil, is a pre-requisite for the domestication of a species8

. Initially, seedling­derived callus grew slowly, producing somatic embryos and non-embryogenic callus (Fig. 1). Growth of the cultures was slow, thereby, acting as a limiting factor to carry out detailed investigation. Therefore, medium salts were manipulated to op­timize the growth and embryogenesis in the cul­tures.

Effect of inorganic nitrogen source---Somatic embryogenesis and callus growth obtained with varied nitrate and ammonium nitrogen are pre­sented in Table 1. Growth of the tissues was al­most same on most of the treatments except those of very high nitrogen levels. Overall nitrogen re­quirement was lower than that of standard MS me­dium (i.e., total 18.40 mgNL-I

). Higher concentra­tions of nitrogen were inhibitory to growth and embryogenesis, irrespective of the type of nitro­gen. Maximum number of somatic embryos (28Iinoculum) were observed on the medium con­taining 50 mgNL- ' (NH4)2S0. (ammonium nitro­gen) and 200 mgNL-1 KN03 (nitrate nitrogen) but, the relative number of somatic embryos produced per fresh weight of the cultures on this medium was lower than that on the medium containing 100 mgNL-' (NH4)2Sc)4 and 400 mgNL-' KN03• These two media (referred to as MS-2 and MS-9, respec­tively) were selected, tissues were grown for 4

subcultures and MS-9 medium was finally used as stock maintenance medium as non-embryogenic callus increased on MS-2 medium during growth in passages. No embryo fonnation was observed in the tissues grown on the medium containing higher concentration of KN03 (300 mgNL-') and (NH.)2S0. (500 mgNL-').

It may be concluded that ammonium to nitrate nitrogen in the ratio of 1:4 or 1:5 at low total nitro­gen level in the medium was favourable for growth and somatic embryogenesis in C. borivilianum.

Effect of plant growth regulators-The results obtained with the effect of varied concentrations and combinations of 2,4-D and various cytokinins viz., BAP, 2-iP, kinetin, TDZ are presented in Ta­ble 2. Maximum number of somatic embryos (24Iinoculum) were obtained on MS-9 medium containing 1.12 )lM 2,4-D and 7.42 )lM 2-iP. Higher concentrations of 2,4-D (~ 2.25 )lM) in the medium inhibited somatic embryogenesis. It was concluded that 2,4-D at lower concentration (1.12 )lM) was beneficial for somatic embryogenesis. On the basis of the number of somatic embryos pro­duced, growth of cultures, colour and texture of tissues, effectiveness of different cytokinins in de­creasing order was 2-iP > TDZ >kinetin >BAP. Therefore, 2-iP was used in subsequent experi­ments.

Effect of amino acids-Incorporation of glutamine or proline (10-400 mgL") in MS-9 medium had a stimulatory effect on somatic embryogenesis and growth of the cultures (Table 3). Maximum number of somatic embryos (63Iinoculum) were obtained on the medium containing 100 mgL-' proline, which was 2.6-folds higher than the control. The medium containing 100 mgL-' proline, 7.42 )lM 2-iP and 1.12 )lM 2,4-D was optimal for somatic embryogenesis. Incorporation of glutamine in the medium had no marked effect on somatic embryogenesis and growth of the cultures.

Long-tenn maintenance (more than 6 months) of embryogenic cultures on the optimal medium resulted in the fonnation of abnonnal somatic embryos and non-embryogenic tissue increased more vigorously than slow growing embryogenic tissues. Such cultures produced globular embryo­like structures (Fig. 3) on MS-9 medium. Proportion of non-embryogenic tissue increased

78 INDIAN 1. EXP. BIOL., JANUARY 1999

Fig. I-Embryogenic (E) and non-embryogenic (NE) callus initiated from seedling. Fig. 2-Isolated embryogenic callus showing a few germinating embryos at 4-weeks growth on MS-9 medium. Fig. 3--One-year-old cultures turned non-embryo,genic and showing globular embryo-like structures. Fig. 4--On transfer to germinating medium, old cultures produced abnormal embryos which did not germinate, instead produced roots or callus. Fig. 5-Isolated somatic embryos germinating on MS-9 medium containing BAP. Fig. 6--Well developed juvenile plantlets (8-weeks old) grown on MS-9 medium supplemented with \3.2 JlMBAP.

ARORA et al. : SOMATIC EMBRYOGENESIS IN CHLOROPHYTUM 79

Table I-Effect of dl1Terent concentrations and combinations of KN03 and (NH')2S0. incorporated in MS medium supple-mented with 2.25 ~ 2,4-D and 1.15 ~ kinetin.

(NH')2S0• KN03(mg N Lot) 100 200 300 400 500

(mg N Lot )

50 SEt SD ;) 28.0 ± 2.7 19.2 ± 2.9 7.0 ± 1.0 7.6 ± l.l GI 3.33 4.82 3.34 3.42 3.54

100 SE t SD 21.0 ± 3.1 18.0 ± 2.5 10.0 ± \. 6 22.0 ± 2.6 2 1.0 ± \. 6 GI 3.44 3.34 3.35 3.14 3.47

200 SE±SD 18.4 ± 2.7 19.6± \,7 19.2±1.3 20.0 ± 2.6 17.4±1.5 GI 3.55 3.54 3.45 3.4 1 3.13

300 SE±SD 9.0 ± \,2 17.8 ± 2.1 20.0± 2.1 7.2 ± 0.8 0 GI 2.05 3.54 3.46 1.9 2.0

Table 2-Effect of different concentrations and combinations of 2,4-D and various cytokinins incorporated in MS-9 medium.

Cytok i,,:n 2,4-D (~ (~ BA P \.10 SEtSD

01 2.20 SEtSO

GI 4.40 SEtSD

GI

2-iP 1.48 SEtSD GI

2.97 SEtSD J

7.42 SE±SD GI

K' nctin 1. 15 SE±SD GI

2.30 SE±SD GI

4.60 SE±SD GI

TDZ 0.45 SE±SD G!

1. 13 SE±SD GI

2.27 SE±SD GI

'li th th increase in age of the culnlres . Thus, embryogenic potential was difficult to maintain in

ong-tenn cultures. Therefore, isolation of fre h culture' from shoot cultures was attempted to have cn hnuous supply of unifonn responding cultures vi thout age effect. Cultures were discarded after 6

months growth. Cyto logical changes associated with various types of cultures were also attempted.

Somatic embryogenesis fr0 1l/ stem-disc derived

cullus--Callus obtained from stem-disc derived

1. 12 2.25 4.50

5.0 ± \.0 14.3 ± 2.0 9.3 ± 0.6

2.06 2.02 2.04 2.0 ± 1.0 10.6 ± 1.5 4.6 ± 0.6

2.07 2.04 2.08 8.0 ± 1.0 11.3 ± 1.5 13 .3 ± 0.6

2.29 2.09 2.09

9.0 ± 1.0 14 .6 ± 2.5 13.2 ± I. 2.24 2.06 2.67

13.6 ± 1.5 9.66 ± 1.5 13.3 ± 2.0 4. 26 2.03 2.03

24 .3 ± 3.0 20.3 ± 2.5 20. 1 ± 2.0 3.55 2.19 204

2 .3 ± 1.3 4.6 ± 0.6 15.6± \.2 3.44 1.92 1.60

22.0 ±2.6 11.0 ± 0.8 I\.6 ± I 5 .3 .38 1.90 \.9 j

15 .6±1.1 12.5 ± 2.0 14.6 ± I.I \.9.3 1.90 1.59

'J .0 ± 1.3 10.0 ± LO 10.0 ± 0.9 ' "t ~) ._J .I. 1.98 190

15.0 ± 2.0 20.0 ± 1.0 11.3 ± 0.6 1.6) 3.42 1.90

10.0 ± 1.7 :;1.0 ± 1.7 12.3 ± 1.5 1.94 3.51 1.9 1

shoot cultures of C. borivilianum on B5 mediu n supplemented wi th 22 ~ TDZ and 0 .1 8 rnM adenine was light green in colour, amorphous and fragi le l3 . Such callus produced large number of somatic embryos when transferred onto MS-9 medium containing 1.12 ~ 2,4-D, 7.42 ~ 2-iP and 100 mgLo' proline. After 3 weeks growth of somatic embryos on the main tenance medium, green tips were observed. During fourth week a few somatic embryos started genninating. All such

80 INDIAN J. EXP. BIOL. , JANUARY 1999

Table 3-Effect of di fferent concentrations of different amino ac ids incorporated in MS-9 medium supplemented

with 1.12 ~M 2,4-0 and 7.42 ~M 2-j.P.

Amino acids (mgL" )

Control

Glutamine 10 50 100 200 400

10 50 100 200

No. of somatic Growth embryos ± SO Index

24 .0 ± 4.2 3.66

24 .0 ± 2.9 3.72 24.5 ± 3.4 3.64 26.2 ± 2.1 3.68 27.5 ± 3.5 3.69 20. 1 ± \.8 3.50

29.2 ± 4.3 4.11 58.5 ± 3.1 3.73 62 .7 ± 2.2 379 53 .7 ± 1.5 3.69

material in tenns of vigour and yield. After over­wintering, plants were sprouted in the next year.

We report here several folds multiplication rate which was advantageous than three-folds multipli­cation rate, a minimum requirement for commer­cial propagation programme l9

. Starting from 10 g fresh cytologically-stable callus, 1355 plantlets were successfully transferred in the field in 2 months.

Unstability and decline in embryogenic potential was major/main problem with the seedling derived callus for long-tenn maintenance. This problem has overcome by using callus obtained from shoot fonning cultures maintained separately. This fonns a cyclic system constitute of shoots -» callus -» embryogenesis -» genninated

-------------------- embryos -» plantlet -» callus. 400 44.7 ± 3.4 3.68

embryos were transferred onto the gennination medium. Maintenance of cytological stability in the cultures is a pre-requisite for obtaining unifonn population of plantlets. Fresh embryogenic culhlres raised through stem-disc derived callus were cytologically stable.

Germination of somatic embryos and transfer to

soil--8omatic embryos isolated from old (more than 6 months) seedling cultures failed to genninate in to complete plantlet on all the treatments tested but, showed precocIOUS gennination because fast growing non­embryogenic cultures outgrowth the embryos. On the other hand, somatic embryos isolated from 1-4 months-old stem-disc/seedling derived cultures genninated into complete plantlet on transfer to BAP (13 .2-30.8 IJM) containing medium (Table 4) within 2 weeks time (Fig. 5). Gemlination of somatic embryos was maximum (48%) on the medium containing 13.2 ~ BAP (Fig. 6). Higher concentration of BAP (30.8 IJM) in the medium completely inhibited embryo gennination.

Plantlets developed through gennination of somatic embryos were successfully transferred in soil with a high rate of survivability (90%). Starting from 109 fresh stem-disc derived callus, 3135 somatic embryos could produced, of which 1505 embryos were genninated in vitro into plantlets and 1355 plantlets were successfully transferred in the soil. ?Iants developed through somatic embryogenesis were comparable to in vivo

Cy tology--Cytological preparations of root tip squashes of gemlinated somatic embryos showed a

Table 4--Germination of somatic embryos isolated from I­S months old cultures on MS-9 medium.

Medium

Control

BAP (~ 13 .2

17.6 22 .0 26 .4 30.8

No. of embryo germinatedJ25 embryos ± SD (%)

0.6± \.15 (2.7) 3.6 ± 0.8 (15 .2)

12.0 ± 2.4 (48.0) 8.0 ± 2.3 (33.3) 3.3 ± 0.5 (13 .8)

Nii

Table 5--{;hromosome numbers of germinated somatic embryos of C. borivilianum showing different levels of

ploidy.

Material

A. Roots: In vitro plantlets*

B. Roots: Normal em­bryos

C. Roots: Abnormal embryos in 6-8 months-old cultures

O. Roots: Abnormal (!mbryos in \8-24 months-old cultures

Chromosome Ploidy level number

24 3X

21 to 27 3X-3 to 3X+3

23 to 36 3X-I to 4X+4

39 to 58 5X-\ to 7X+2

·Plantlets raised through organogenesis from stem-disc explants U

ARORA et al.: SOMATIC EMBRYOGENESIS IN CHLOROPHYTUM 81

Figs 7 & 8-lncreased ploidy was associated with abnormal embryos. Root tip squashes of abnonnal embryos showing 51 (Fig. 7) and 58 chromosomes (Fig. 8).

wide variation in chromosome numbers (Table 5). A clear tendency of increase in ploidy level during in vitro growth of cultures is evident from the results. Low cytological variation (3X-I to 3X + 3) was observed in the embryos germinated in 1 to 4 months-old cultures. Somatic cell chromosome number iri C. borivilianum is 2n=1620

, while in other related species it is 2n=28, 32,40,42, 8421

,22.

Thus, there are two basic chromosome number in the genus Chlorophytum, viz. n=7 and the other being n=8. Precociously germinated somatic embryos in 6-8 and 18-24 months-old cultures showed a wide range of variation in the ploidy level, ranging from 3X-3 to 4X+4 and 5X+I to 7X+2, respectively (Figs 6 & 7). Therefore, these somatic embryos were supposed to be abnormal and thus failed to germinate into a complete plantlet. More cytological work is required to know the chromosome behaviour in field grown plants and cause of poor seed set.

Ramawat. Authors are also thankful to Dr. R.C. Verma, Department of Botany, Vikram University, Ujjain for his help in cytology of th~ material.

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Experiments are in progress to develop a tech­nology for in vitro production of mass saplings of 8

C. borivilianum using a bio-reactor system for its domestication.

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Acknowledgement

This work was supported by grant-in-aid for re­search from the leAR, New Delhi, to Dr. K.G.

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