Indian Jou rnal of Experimenta l Biology Vol. 4 1, November 2003. pp. 13 11 - 13 16

In vitro control of fasciation in proliferating nucel1ar embryos of Mangifera indica L. var totapari red small for cloning

* H C Chaturvedi , S Agnihotri. M Sharllla, A K Sharma. M Jain & A Chouras ia

Tissue Cul ture Laboratory. ati ona l Botanica l Research Institute, Lucknow 226 00 I . Inuia

I?ece i" ed 22 lIeril 2003; revised 30 M({v 2003

Nucellar ti ss ue contained in ovular hal ves of young frui ts of MOllgilera illdica L. totapari red small. a dwarfing rootstock. differentiated faseiateu embryonal structures in presence of 6-benzy laminopurine II3A P(O.15 mg 1" ) 1. 6-(y-y­dimeth y lall y lamino) purine 12iP(O.15 mg 1'1) 1 ;l1ld indole-3-aecti c acid r(IAA(O.5 mg 1' 1) 1 incorpora ted in th e semiso lid medium during 50-60 days. Due to embryona l fasc iation. hard ly 2-3 well - formeu embryos cuuld be obta ined pCI' culture of proliferat ing embryos. Of the 3 etby lene inhibitors I L-a-(2-aminoethoxyv iny l)-glyc ine-HCI (A VG) , AgNO, and salicy li c acid (SA) lused, embryonal fasc iation and necrosis of intervening ti ssue was eO lllpletely control led by 3-4 subcultures of ra sc iated mass of cmbryos under the inlluenee of AVG (0.05 Illg 1,1) in presence or auen ine su lphate lAdS (SO Il1g 1' 1) 1 incorporatcd in the same medium. A lmost synchroni zed development or isolated embryos. measurill g ca 2 cm in length . wa, obsc rved in a different medium used in liqu id stationary state and supplemented. parti cularl y w ith stress-produc ing substances l absc isie ac id (ABA . 0.0 1 mg 1,1) : and pol yethy lene glyco l (PEG. 100 mg 1' 1) 1 besides certain uther moui fications. About 34% convertibilit y of processed embryos was obtained during a period of 90 days. The plantlets had well -developed roots along wi th laterals which were longer than leafy shoots. I II "ilro rai sed plants su rvived ex vi lm for about 2 Illonths.

Keywords : ConvertibililY of embryos . Dwarfing rootstock, Eth y lene inhi bitors. Fasc iat ion or embryos. MOlIgij'em illdic(I tOlapari red small. ucc llar embryos

Establi shment of high-density orchards of Mangifera indica L. (mango) compri sing uniform trees warrants ava ilability of clonal pl ants of dwarfing rootstocks. Such a useful proposition is virtuall y not poss ible through conventi onal methods of propagation , particul arl y in mango being a highly heterozygous tree and most intractable to be vegetati vely propagated I . Even if the stem cuttings are rooted, alheit with a very low percentage, the adventitious roots are undesirable in case of trees, sin ce such roots provide poor anchorage to them". That is why , the cloning of mango through nucell ar embryos is of great signifi cance mainly beca use of ava ilability of ready-made root system in them and their hi gh rate of mu ltipli cationJ

-6 However, despite an ex tensive

research done in thi s field for more than a decade, success is not yet to be achieved due to poor and unpredi ctable convertibility (plantlet formation) of nucellar embryos in vitro and virtuall y no success of

I 4 transp ant .

"'Correspondent au thor Phone: (+9 1 522) 2205840 Fax: (+9 1 522) 2205836 E-mai l : hec_nbri @hotmai l.com : hcc_ vishram@yahoo.co. in

We now report advances made tn rap id proliferation of nucell ar embryos of M. indica var totapari red small. a dwarfing rootstock . without intervening callus phase, their almost sy nchroni zed development and production of normal-look ing plantlets with a good root system and their ex vitro­growth .

Materials and Methods Young fruits of 25-30 days of age, measuring ca

1.5-2.5 cm in length , were coll ected from a tree of Mang(/era indica L. val' totapari red small - a less polyembryonic va riety, grown in an experimental orchard of G.B . Pant University of Agriculture and Tec hnology, Pantnagar. Fruits were first washed in running tap water for 30 min , pretreated with 5% of labolene (G laxo Smith Kline Pharmaceutica ls Ltd ., Mumbai, India) so lution for 10 min and ethanol (95%) for 2 min followed by surface- sterili zati on by chlorine-saturated water for 40 min. The surface­sterili zed fruits were washed thoroughly with sterile di stilled water and were cut longitudinally into two halves . After removing the zygotic embryo, the ovul ar halves containing nucellus ti ssue were scooped out and cultured with the cut face away from the agarified

11 12 INDIAN J EXP 1310L. NOVEMBER 2003

nutrient medium, BM I, formulated for augmenting nucellar embryogenesis. BM I comprised (concentra­ti ons in mg rl) H4N03 (LOOO) , K 0 3 (1500), Ca(N03h (200), (N H4h S04 (100), MgS04 (360), K2S04 (200), KH 2P04 (150), CaCI] (400) , di sodium­ferric-ethylene-diaminetetra-acetate, rNa-Fe-EDT A (5 1111 r l), prepared by dissolving FeS04.7H20 (557 mg) in 100 ml warm solution containing NarEDT A (745 mg) in double di stilled water], trace elements of Murashi ge and Skoog medium 7

,

thiamine- HCI (0.2); pyridoxine-HCI (0. 1), nicotinic acid (0.5) , m-inositol ( 100), glycine (3), L-arginine (1 5), L-glutamine (100), L-asparagine (15 ), ascorbic ac id (25), casein hydrolysate [CH (200YI, sucrose (50,000), and agar powder r(7500), HiMedia Laboratories Ltd. , Mumbai , Indial- It was supple­mented wi th 6-benzylaminopurine !BAP (0. 15 mg r l )l, 6-(y-y-d imethylallylamino) purine [2iP (0. 15 mg rl)), indole-3-acetic ac id [IAA (0.5 mg rl)l and adenine sulphate [AdS ( 15 mg r l)] at initi al stage and for subsequent proliferation of nucellar embryos, the same medium with an increased concentration of AdS, i.e., SO mg r l was employed and named BM2. The embryo proliferation medium, BM2 was suppl emented with 3 ethylene inhibitors at different concentrations, viz., 0.0 I, 0.025, 0.05 and 0.075 L-a­(2-aminoethoxyvinyl)-glycine-HCI (A VG), 5, 10 and 15 mg r l salicylic acid (SA) and 0.1, 0.25 and 0.5 mg rl AgN0 3 in order to see their effect on alleviating the problem of fasciation of embryos and thus, leading to embryo-to-embryo multiplication.

The nutri ent med ium, BM3 used in liquid state for sy nchroni zed development, maturation and convert­ibility of processed nucellar embryos was a modifica­ti on of BM I, containing a reduced concentration of

H4 0 3 (250 mg rl ) and of IAA (0. 1 mg rl) and absence of L-glutamine, CH, BAP and 2iP and having supplement of polyethylene glycol (PEG; 100 mg rl ) and absc isic acid (A BA; 0.0 1 mg rl ). These particular substances and their concentrati ons were se lected on the basis of the best responses obtained in case of other varieties of M. indica already investigated earlier8 in thi s Laboratory.

All the med ia were adjusted to pH 5.8, before adding agar (7500 mg r '), and sterili zed by aUlOclaving at 1.08 kg/c m2 for 15 min . Cultures were incubated under 25 )..lmol m·2s·1 quantum flux density , except menti oned otherwise, for 15 hr a day at 27° ± 1°C and RH 70 ± 5%.

The plantlets resulting from the processed nucell ar embryos were acclimati zed ex vitro after transplanta-

tion to potted soilrite. During the process, for hardening of plantlets, the humidity was controlled from high to gradually low, for a period of ca 15 days, by using glass cy linders of 6 cm diam of 3 different lengths (10, 15 and 20 cm) and by placing the lids on them for specific periods following the procedure developed for hardening the in vitro raised jojoba plantletsl). During transplantation , the root system of plantlets was thoroughly washed with sterili zed water and dusted with powder comprising a mixture ( I: I ) of bavistin (BASF, India Limited, MUlllbai ) + nebasulf (Pfizer Limited, Navi Mumbai ). The pOlled plants were initially kept under hi gh light intensity (ca. 37.5 /-lmol m·2s·1 quantum flux density) under controlled conditions of culture room (at 27° ± 1°C and RH 70±5%). Fifty potted plants were subseq uently transferred to glasshouse and finally to field conditions at different times of the year.

Results and Discussion The nucellar ti ssue contained in ovul ar halves

turned brown and remained moribund for ca 30 days followed by meristematic activity in disc rete patches where embryogen ic structures spari ngly di fferentiated associated with ti ssue necrosi s after a further period of 20 to 30 days in BM I . On subculture, such structures gave an appearance of aggregation of embryos, which infact was largely due to pluricotyly and non-separation of semi-differentiated embryos, resulting into a fasciated mass of embryos, which hardly offered 2-3 embryos per culture. Fasciation of embryos is also disadvantageous like necrosis of embryogenic ti ssue, which is generall y encountered in mango nucellar embryogenesis in vitro, greatly

.. h fl · I· . 10 II restricting t e rate 0 mu IIp Icatlon . . Of the three anti ethylene substan ces used , A VG

was most potent to check fasciation of embryos and promote embryo- to-embryo proli feration foll owed by Ag 0 3, while SA was completely ineffec ti ve and on the contrary promoted browning of cultures (Tab le I). Furthermore, A VG was most effec tive at a lower concentrati on (0.05 mg r I) as compared to Ag 0 3,

which even at a 5 times higher concentration (0.25 mg rl ) produced one third of the response obtained with A VG. Also, there was comparatively no browning of cultu res and the embryos produced were also greeni sh and heallhy in A VG-treatment. On the contrary, at optimum concentration of AgN03, the cultures showed browning associated wi th formation of slightly vitrified and abnormal-looking embryos. Such observati ons support the fact that efficacy of

CHATURV EDI el al.: CONTROL OF FASCIATION IN PROLI FERATING EMBRYOS OF MANGO 13 13

A VG in countering ethylene formation in cultures is higher than any other ethylene inhibitors in vogue3

.

In conformjty with the present observations assigning the role of causing embryonal fasciation to high concentration of ethylene, the presence of A VG at its optimum concentration promoted the formation of well­differentiated and normal-looking embryos with suppression of ethylene formation. Due to fasciation, production of number of individual embryos per culture that could be convet1ed into plantlets was reduced even to nil , which indeed is very disadvantageous for clonal multiplication through nucellar embryogenesis. How­ever, A VG too at its higher concentration (0. 1 mg 1' 1), inhibited differentiation of new embryos and also the cultures developed browning of embryogenic ti ssue. The reported stimulation of embryogenesis in cultures of M. indica var tutehau, a polyembryonic variety, by reducing ethylene biosynthesis with A VG 12 is in accordance with the present observation , as also the suppression of proliferation of embryos at its higher concentration through inhibition of spermidine synthesis. The polyamine, spermidine, has a role to play in somatic embryogenesis, its induction as well as embryonal proliferation. which have also been demonstrated in vitro in nucellar explants of mango l2

. Attainment of normal embryogenesis with differentiation of well­organized embryos during the process of their proliferation with the use of A VG and to some extent also AgN03 substantiates that ethylene induces abnormalities during the process of differentiation of regenerants ill vitro as obtained in the present study in the form of fasciat ion of embryoids I3

.14

. Ethylene accumulation has been found to produce vitrification, flaccidity and other growth abnormalities observed in regenerants formed in cultures, particularly in prolonged cultures as shown in case of microshoots of potato, which have been shown to become stoloneferous with smail leavesIS

.16

. Even the shoots of potato grown in culture get abnormal in presence of ethylene accumulation in the culture vessels by becoming stoloneferous with small leaves.

In the initial morphogenetic treatment, hardly 2-3 well-formed embryos could be separated from apparently normal-looking groups of embryos due to excessive fasciation accompanied by necrosis of intervening tissue amongst proli ferating embryos as also the young embryos during the advancing culture period (Fig. I ). After an average of 3 subcultures in the morphogenetic medium supplemented with A VG (0.05 mg 1' 1), as many as an average of 2 1 normal and healthy embryos could be retrieved (Table I ), and after

subsequent subcultures in the same treatment norma l proliferation of embryos without fasciation was obtained (Fig. 2).

Unlike other mango varieties, sporadic plantlet formation from amongst the embryos proliferating on nutrient agar was absent in cultures of totapari red small , albeit some of the developed embryos did form roots. However, ca 52% germinated embryos. i.e., having visible plumule and developed root were obtained after incubation for 60 days. when the somatic embryos at the cotyledonary stage measuring ca 2 cm in length were cultured in the liquid medium 8M3. Responses of cotyledonary nucellar embryos of 4 sizes (ca 0.8, I , 1.5 and 2 cm in length) are given in Table 2. In the liquid stationary state of nutrient medium, almost synchronized development of embryos was seen (Fig. 3). Percentage of embryos attaining maturat ion , germination and conversion stages were directly proportional to size of embryos. Whi 1st, the smallest embryos did neither show germinati on nor conversion. the biggest embryos showed ca 34% convers ion into plant lets. Furthermore. all the embryos, which appeared mature, hav ing a developed root, though appeared similar, did not have the same potentiality to form plantlets. For example, embryos of the opti mum developmental tage on being reared in the cul ture medium for a period of 40 days showed 80% maturation. of which 52 0/£ germinated during a further period of 20 days, while 34 0/£ of the germinated embryos on subculture in the same medium were actuall y converted into plantlets during a period of 20-30 days. It appears that A VG, besides controlling fasciation and other abnormalities of embryos also promoted their potentiality to form plantlets. since the normal-look ing developed embryos. isolated from cultures of embryonal masses though grew in size with abnormally massi ve coty ledons during processi ng ill 8M3 rarely converted into plant lets. In contrast. their counterparts differentiated in the presence of A VG developed into normal plantlets III fairly good percentage. This effect of A VG has rarely been reported, but suppression of ethy lene production by anionic complex of sil vet1hiosulphate [Ag(S}O:;)" I has been shown to promote regenerat ion of plant lets from lea f explants of potato cultured in vitro l7. It may be difficult to assign exact reason fo r A VG-mediatcd enhanced potentiality of embryos to develop into plantlets. but elimination of senesc ing influence of ethy lene during histo-differentiation and development of embryos can be expected to play a role in th is contex t.

1314 INDIAN J EX P BIOL, NOVEMBER 20m

The proccssed embryos which had initi al ly massive '\lhite cotyledons :.ind black mots, turned green hefore gcnnin<lti,)n anu producing normal he,t1th y looking pl anrlets havi ng proportionate roots wi th latera !s (Fig . ..J.). Nci ther dun n1,\ prol i ferati on or n ucellar cm­bryos 11l)r during the ucvc lopll1cntai ph ase or isolated nucc llar embryos vitri f ication or hyperh ydri city, which is reponed to be or C,)111 111011 "ccurrc nce in

plantlct regcneration through nll cell ar ell1bry ,)gc lles is . IS IC' . d ' h d In 111 ;:\ngo ' was wltncsse 111 r c prescnt stu y. Also, there was nl) intervening callusing or abnormal ti ssue formlltioll at the juncture of shoot and root sys· tem of plant lets . Such results may he as~ i g ll ed to a strategy di f ferent f rol11 that followed earl ier, i. e., ill­duction of embryu differentiation in ~. u s pe l\ sion cu l-

. II I I ) t) - 00 tun: oj nuce aT callus .- --. In thc pres nt study_

1-'i)C" 1-5 - CUltUIT\ or MCll1gij, 'w iI/dim ,'ar t')I~p"r; red sma ll nllccl iar emhryogcnc\i,. ( I ) Init i::! , tage or prolircr .. liion or cmbryo;, ,hml ing cmhryonal r;.scia lion and necros; , . X I .~: (2) A llcl illtiun ur cmhryonal rasc ialiun and ncci'l), is in a cul tu rc t)f proliferating C IIl ­

bryo,. X 1.-1 : (3) Almost "Y!1Chron izcd (i.:\'clopll1ent or cmbryos . X 1.-17: (-I ) Convert ibilit y (plantl ct r.)!'I l1'llion) or processcd cmbryos. X I.:::: and (5) A,l ill I·if!'(' r~li,ed plan llel )!nlwing in soilritc. X 1.07.

...

CHATURVEDI el 01.: CONTROL O F FASCIATION IN PROLIFERATING EMBRYOS OF MANGO 1315

Tab le I - Effec ts of some antiethylene substances on a llev iati on of fasciation during proliferation of nuce ll ar embryos of M. indica L. var to tapari red small

I Values are mean ± SE of 5 replicatio ns]

rreat ment Extent of : onc . mg rl fasc iati on

No. of indi vidual embryos per c ulture*

Remarks

: ontrol ++++ 2A ±0.25 Fasc iated mass of embryonal structures

\ VG 001 ++++ 4 .8 ±0.87

12.4± 1.1 3

2 1.6± 1.45

2.8±0.34

Very lillIe embryonal proliferati on ).025

).05 ++ nil

Embryonal proliferation, a few whitish e mbryos

Hea lth y green/ white shapely embryos ).075 + + 1.10 + ++ nil

Poor e mbryonal prolife ration, root formation in embryos

Necrosis of cu ltures

\ gNOJ O.1 + ++ 6.4±0.68

7.4± 1.02

3.0±0.60

Vitrified, slightly abnormal embryos

1.25

0.5 ++ + +

LillI e embryonal proliferation, some vitrified embryos , sli ght necros is of cultures

Necrosis of cultures, lillIe embryonal proliferation

;A 5 ++++ nil

nil

nil

Necrosis o f cultures

0

5 ++++ ++++

Necrosis of cu ltures

Excessive necrosis o f cultures

~ul1lber of + marks connotes the ex tent o f response 'After incubation for 60 days

Table 2 - Effec ts o f differelll developmental stages of nuce llar embryos (NE) of M. indica var totapa ri red small on development , maturati on. germination and convertibility in medi um BM 3

rValues are mean ± SE of 10 replications l

Initi a l stage o f Length of inoculated Deve lopment Maturation Germination Conversion inoculated NE (Le ngth of NE afte r (Deve loped root (V is ible plumule (Plant le ts from

coty ledonary NE (e m) 20 days) format ion afte r 40 a long with germinated NE (cm) days) developed root after after

(%) 60 days) 30 days) (%) (%)

0.79±0.02 1.52±0. 13 20±5 . 17 8±3.27 2.4 ± 0.35 II 1.0 ±0.04 2.54±0.06 56±6.53 18 ± 4 .66 6 ± 3.06 III 1.5 ±0. 16 3.05±0.06 64±4.0 26±4.27 10±3.33 IV 2.01 ±0.08 4 .08±0.07 80±3.65 52±3.75 34±2A5

Size of coty ledonary nucell ar e mbryos - (1)-0.8 ; (11 )- 1: (111 )- 1.5: and (lV)-2 cm in length

nucellar embryos were differentiated from the nuce ll us ti ssue contained in ovular halves without the intervening callus phase and on a semisolid medium. Well­differentiated embryos of cotyledonary stage were proc­essed for convel1ibility in the liquid state of the medium . Presence of ABA , a growth inhibitor in the medium as also of a high osmoticum creating substance, viz., PEG also contributed to limit both hypel1rophy of cells and callus formation by creating stress condition.

Of the different pe ri ods, during which the ill vilro­

raised pl antlets were transpl anted to so il , co mparati vely bette r results were obtained during

rainy season (at 30°-35°C and RH 85-95%) with 80 to 90% transplant success, while the potted plants a lso pi cked up growth and appeared normal (Fig . 5). However, the unpredictable convertibi lity of

otherwise perfectly normal-looking embryos, which have well-developed root system and even a meri stematica lly activated plumule is one of the big snags in clonal multiplication o f mango through in vilro nueellar e mbryogenesis, which has a lso been experienced by several other workers to di fferent extents I 1.2 1. The problem is still worst confounded with the hi gh rate of morta lity of apparently healthy look ing in vilro ra ised pl antlets after giving a hi gh transpl ant success. The majority of such pl antlets gradual ly e ither developed blackening of stem beg inning from the base at the level of the cotyledons and spread ing up-wards or the shoot tip developed necrosis, while still in other eases the older leaves showed necrosis at tips after ca 2 month s of growth in the potted soil and ultimate ly led to death of plantlets.

1316 INDIAN J EXP BIOL, NOVEMBER 2003

Acknowledgement The authors, H C C and S A, thank CS IR, New

Delhi, for granting Emeritus Scientist and RA , respectively, while all the authors also thank DBT,

ew Del hi , for fi nancial ass istance. Thanks are al so due to the Director, NBRI, Lucknow, for faci lities provided and to Professor S R Yadav, G.B. Pant Uni vers ity of Agriculture and Technology, Pantnagar, India for prov iding young fru its of mango totapari red sillall .

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