Indian Journal of Experimental Biology Vol. 37, January 1999, pp: 70-74

Repetitive somatic embryogenesis in aggregated liquid culture of Bunium persicum Boiss

S Grewal & Manju Rani

Regional Research Laboratory, Canal Road, Jammu 180001 , India

Received 29 May 1998; revised 9 September 1998

Conditions for regeneration of B. persicum plants via somatic embryogenesis in aggregated liquid cultures were defmed. Globular somatic embryos with extended radicals were isolated. These embryos remained arrested at this stage in liquid medium and when cultured for a longer period, secondary em­bryonic masses developed from the shoot pole. This repetitive embryogenesis continued until trans­ferred to agar medium for further development of embryos and ~ubsequent conversion into pIa; ,lets. Arrested somatic embryos from aggregated liquid cultures have a commercial potential and p 'esent findings could provide a base for exploitation.

Bimium persicum seeds (kalazira) are ruthlessly collected from the wild, the only source, thus placing this plant species under endangered cate­gory. The plants can be propagated by seeds or vegetatively from tubers. Three to four year juve­nile period is required rot 11 seed to seed cycle to develop into kalazira tuber necessary for planting in the field . Long juvenile period to get mature tubers for field cultivation has hampered the con­ventional cultivation and breeding programmes and inspite of high material costs, systematic culti­vation is not undertaken anywhere. Plant tissue and cell culture can successfully be used as an alterna­tive to produce propagules for field delivery. Al­though somatic embryogenesis 1.2 and organogene­sis2 in callus cultures of B. persicum are on record, the studies regarding embryogenesis in liquid cul­tures of B. persicum have not been attended to. Pre­sent study therefore, is aimed in this direction and describes embryogenic aggregated liquid cultures and conversion of somatic embryos into plantlets.

Materials and Methods Initiation, proliferation and maintenance of

embryogenic liquid cultures. The embryogenic calli used to initiate liquid cultures were taken from mucilaginous, granular, pale yellow calli obtained from leaf discs as described earlier. One week old callus (1.0 g wet) was transferred to Erlenmeyer flasks (100 ml) c~ntaining liquid MS medium3

(40 ml) consisting of Kn,(5xlO~M) and 2,4-D,

(1 x I O~ M). These flasks were placed on a rotary shaker (Emenvee) at 90 rpin in dark at 25°±2°C. After four weeks, the entire content of one flask was

. divided into two flasks and equal volumes of fresh medium (20 ml) was added to each flask. After 4 weeks, the culture contained suspension of single cells and aggregate cell masses. The cell aggregates were allowed to settle, medium was decanted and 1 g of tissue was transferred to fresh medium. The stock cultures were maintained for 4 weeks. For experimental purposes settled cell aggregates from 15-daysold cultures were used. Samples of aggregated suspensions were transferred to small Petri dishes and observed under an inverted microscope (Zeiss inverted microscope televel).

Growth kinetics-Growth rate of embryonic masses in liquid medium was recorded by inoculating known amount of tissue from 15 days old stock cultures and grown for 30 days. This was followed by harvesting and reweighing of fresh embryonal masses at 5 day intervals, which were obtained after filteration under vacuum. Embryo formation was determined from five samples (250 mg) taken at random and counted number of embryos formed using a hand lens. Similar procedure was adopted for agar cultures2.Biomass production and embryo formation was compared in both systems.

Maturation and conversion to plantlets--Four week old embryogenic cell aggregates (1 g wet)

GREWAL & RANI: SOMATIC EMBRYOGENESIS OF BUNJUM 71

from liquid cultures were transferred and spread in Erlenmeyer flasks(100 ml) containing MS agar medium (40 ml) and kinetin (5xl0~ M). Cultures were kept under 16 hr photoperiod at 25°±2°C. pH was always adjusted to 5.8 prior to outoclaving at 121°C for 15 min. Five replicates were used for each treatment.

Results and Discussion

Embryogenic calli after transfer to MS liquid medium supplemented with Kn, (5xl0~ M) + 2,4-D, (l x lO-<> M) gradually formed si."lgle cells and cell aggregates (up to 15 mm; Fig. 1 A,B). These were pale yellow, mucilaginous and maintained on above mentioned medium by subculturing after 4 weeks. The growth of cultures was measured after 6 month of culture and compared with agar cultures. Liquid cultures under control conditions showed a sigmoid growth curve and reached a maximum of 12.5 glculture flask after 20 days (Fig. 2A). This value was higher (12.5 fold) than initial inoculum weight

I

and 1.5 time higher than cultures grown on agar medium of similar composItton (Fig. 2B). Microsopic examination of liquid cultures revealed numerous aggregates of somatic embryos (SE). Numbe!" of SE formed in liquid medium was compared with SE formed in agar medium over 30 days culture period. Figs 2 C and D clearly show more SE formation in liquid medium (1.2 times on per g basis and 1.3 times on absolute basis). Liquid cultures ha ve now been maintained for over one year without apparent loss of embryogenic capacity. Somatic embryo development Ylli - i! -ill duration of culture in liquid medium was studied. Globular embryos showed slight elongation with radicle pole extending further when cultured for longer time.The growth of most of the somatic embryos was arrested at this stage (Fig. 1 C). Lack of synchrony is one of the major problems encountered with SE production for commercial realization4

, however, such an arrested SE production in liquid cultures could prove to be commercially interesting in Bunium persicum system. But, culturing for longer periods in this medium (6 weeks) resulted in root development. The shoot pole callused and subsequently secondary embryonic masses developed (Fig. 1 C). This repetitive embryogenesis continued till transfer of suspensions to agar medium for further development. Mature e!-llbryos

(embryos with distinct bipolar structures) were observed sporadically (Fig. ID) in liquid medium. These embryos were generally vitrified, a physiological disorde~ also seen in soybean6 and peanue embryogenic cultures in liquid medium. Vitrification could be a serious impediment· to the development of liquid culture system in some susceptible plant speciess. At the same time, it is a phenomenon that may be useful in micropropagation of some other species, due to its capacity to boost multiplication rates8

•

Germination and conversion into

plantlets--Solid medium was required for further development of globular embryos and conversion into plantlets. Aggregated embryonic masses were transferred to agar medium containing Kn (1 x I O~ M) alone. After a latent phase of one week, cultures showed proliferation of callus. This was probably due to the carry over effect of 2,4-D. After 1-2 months, embryos at all stages of development (globular, heart, torpedo etc.; Fig. IF), together with plantlets were observed in the same medium (Fig. IE,G). Embryos in agar medium developed as--{I) root was formed, where shoot pole development was inhibited (Fig. I H); (2) shoot and perhaps then root was formed (Fig. 11); and (3) simultaneous development of shoot and root pole were observed (Fig. 11).

Inhibition of shoot pole development by early root formation is a problem encountered in several other plant tissue culture systems9012

• The plantlets of different sizes were easily separable from one another and had developed root, shoot and glabrous tuber at their junction. At this point, efficiency of plant regeneration was also recorded. After 10 week incubation, shoot growth was · etiolated with rudimentary leaves, upright thin stem and microtuber at the base (Table 1). MS- medium at half- strength constituents induced more conversion of somatic embryos with marginally longer and more tubering shoots. However, tuber size was one third of full-strength medium grown tubers. In nature seeds germinate in 3-4 months after passi~g through winter of that year and produce only a couple of leaves. The plant dies leaving small tuber 13. The first year grown plants from seeds in nature were compared for small tuber formation with etiolated plants having microtubers obtained in vitro from somatic embryos in the present study.

72 INDIAN J. EXP. BIOL., JANUARY 1999

The results from present study therefore suggest that MS medium with 2,4 -D and Kn were essential for establishment and maintenance of embryogenic competence of aggregated liquid cultures. Agar medium supplemented with Kn alone was required

A

c

'F G

for germination of embryos and · their conversion into plantlets. Thus, establishment of repetitively embryogenic cultures in liquid medium extends the information previously available on Bunium persicum somatic embryogenesis. This technique is

B

D E

I ' J

Fig. I-Regeneration iii Bunium persicum from embryogenic aggregated liquid culture. (A, B)-proliferation of embryogenic aggregates in liquid MS medium; (C)-arrested somatic embryos and development of secondary embroynal masses at shoot pole; (D)-vitrified mature somatic embryo in liquid medium; (E,F,G)-various developmental stages of somatic embryos, (F)-plant conversion; (E,G)-in agar medium; (H)-root formation prior to shoot development; (I)-shoot pole formation prior to root· devel­opment; and (J)-simultaneous development of shoot and root pole.

GREWAL & RANI: SOMA TIC EMBRYOGENESIS OF BUNJUM 73

Table l--Effect of MS-medium constituents on efficiency of regeneration and microttiber development in Bunium persicum

[Values are mean ±SE of 5 replication per treatment]

MS-medium Plant conversion Shoot size Tubering shoots Mean tuber fresh wt (%) (cm) (%) (mg)

Full 20± I 3 ± 0.5 30±2 20± 2.5

Half 55 ± 2.5 4 ± 0.45 45 ± 3 6±0.5

Biomass increment in liquid medium Comparison of biomass increment in agar & liquid cjJltures

20 20

--Agor (B) (A)

! ----Liquid ! ::J

E ~ ',}" iJ 0

c, t ' "f " ..... 1: 10 10 i" ----. .2' iii I ! , ,

Ii. , , .c: :I , , II I ! .--1' u. ....

Z" ..

" " " 0 0

0 5 10 15 20 25 30 0 5 10 15 20 25 30

DAYS DAVS

Embryo multiplication in agar and liquid Emb, /0 mUI(iplication in agar and liquid

cultures cultures

50 500

-- Agor (C) --Agor ( D)

40 ---- Liquid 400 ----Liquid }------'" "

.. ! "

.. I .. ""

I .. .!?' " 2 I ..

" , ..

30 :; 300 .. III .. U I .. .. I .. 0 .. I .. ~ .. III I

D 0 I

E ~ I

20 200 I W D I

E I

w ,f "

10 100 , ....

~'

o~--~----~ ____ ~ ____ ~ __ ~ OL-----~--_r~----~------L---~

5 10 15 20 25 30 5 10 20 25 30

DAYS DAYS

Fig. 2~rowth and embryo formation in liquid and agar media. (A)-growth curve in liquid medium; (B)-comparison of growth in liquid and agar media; (C,D)-comparison of embryo formation in liquid and agar media.

74 [NDIAN 1. EXP. BIOL., JANUARY 1999

the first step in the development of large scale Bunium propagation using bioreactors and artificial seed technology.

Acknowledgement The author are thankful to Professor S S Handa,

Director, RRL for extending help. Thanks are also due to Professor C K Atal for helpful discussions and Dr 0 Koul, RRL, Jammu for advice in preparing the manuscript. Thanks are also due to Mr Anokh Singh, RRL, Jammu for photographic work. References I Wakhlu A K, Nagori S & Barna K S, Plant Cell Rep .• 9"

(\991) 137.' 2 Grewal S, Indian J Exp Bioi, 34 (1996) 356. 3 Murashige T & Shoog F, Physio Plant, 15 (\962) 473. 4 Ammirato P V, in Biotechnology in plant sci­

ences--Relevance to agriculture in the eighties, edited by

M Zaitilin, P Day, A Hollander arid C M Williams (Aca­demic Press [nc., San Diego) 1985, 160.

5 Hussey G, in Plant tissue culture and Its agriculture ap­plications. edited by L A Withers and P G Alderson (Butterworths Scientific Publications, London) 1986,69.

6 Finer J J & Nagasawa H, Plant Cell Tissue Organ Cult, 15 (\988) 125.

7 Durham E & R P A Wayne, Plant Cell Rep. II (\992) 122.

8 John A, Forest Research Ed, (\983) 28.

9 Arrillaga I, Brisa M C & Segura J, Plant Cell Rep, 6 (1987) 223.

10 Novak F J, Afza R, Van Duran M, Perea-Dallos M, Con­ger B V & Tang Xiaalong, Biotechnology, 7 (1989) 154.

II Verdeil J L, Buffard-Morel J & Pannetier C, Oleagi Newx, 44 (1989) 403.

12 Touchet B de, Duval V & Panpetier C, Plant Cell Rep, \0 (1991) 529.

13 Munshi A M, Zargar G M, Baba H & Singh T, Indian Hort. 39 (1994) 59.