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High frequency of plant regeneration from hypocotyl


Plant Cell, Tissueand Organ Culture24: 79-82, 1991. ? 1991 KluwerAcademicPublishers. Printedin the Netherlands.

High frequency of plant regeneration from hypocotyl explants of Bras

sica carinata A. Br.
Mei-Zhu Yang, Shi-Rong Jia* & Eng-Chong Pua Institute of Molecular and Cell Biology, National University of Singapore, Kent Ridge, Singapore 0511 (*requests for offprints) Received 12 January 1990; acceptedin revised form 16 September 1990
Key words: Brassica carinata, Ethiopia mustard, hypocotyl, plant regeneration, tissue culture
Abstract

An efficient tissue culture system for high frequency of plant regeneration from hypocotyl explants of Brassica carinata was developed via manipulation of culture medium and selection of explants. Explants grown on medium containing combinations of 2mg 1 ~ BA and 0.01 mg 1 i NAA or 4mg 1 I kinetin and 0.01 mg 1-J 2,4-D regenerated shoots at 100% frequency. High frequency shoot regeneration occurred only from explants originating from 6 to 7-day-old but not younger or older seedlings. Explants showed higher regeneration capacity at the distal end than the proximal end, and the upper segment was more regenerative than the lower segment of hypocotyl. Regenerants were rooted on half-strength growth regulator-free medium, acclimatized and developed into normal, fertile plants.
Abbreviations: BA-benzyladenine, 2-4-D-2,4-dichlorophenoxyacetic acid, NAA-naphthaleneacetic acid, MS-Murashige & Skoog

Introduction

Brassica carinata (Ethiopia mustard), which is extensively cultivated in Africa, posesses several agronomically important traits, e.g. aphid tolerance and disease resistance. This species also has potential to fit rain-fed agriculture conditions because it outyielded the traditional B. juncea (brown mustard) and B. campestris (turnip rape) both in seed and oil yield [1]. In recent years, major efforts in Brassica research have focused on utilization of tissue culture technology for crop improvement. In B. carinata, although plants have been regenerated from cotyledons [7-9], stems [7] and protoplasts [3,12], the frequency of plant regeneration was relatively low. In this communication, we report an efficient tissure culture system for 100% shoot regeneration from hypocotyl explants via selection of explants

and manipulation of growth regulator combinations in culture medium, and subsequent production of normal and fertile plants of B. carinata.

Materials and methods

Seeds of B. carinata accession No. 84A165 were surface-sterilized in 70% ethanol for 30 s, followed by continuous agitation for 30 min in 0.5% sodium hypochlorite (10% (v/v) commercial bleach (Clorox?) solution), then rinsed four times with sterile distilled water. About 20 seeds were germinated on 50ml solidified half-strength MS medium (6) in a GA7 ? container. The container was placed in a tissue culture room under a 16h light/8 h dark cycle with a photon fluence rate of 55-65#mol m 2 S-~ from Sylvania cool white F37T8/CW fluorescent lamps. The day and night

Part of this work was done in BiotechnologyResearch Centre, Chinese Academyof Agricultural Sciences, Beijing 100081,China.

80
Table 1. Effect of BA and NAA combinations on shoot regeneration from upper hypocotyl segments of B. carinata.

NAA (mg 1- i ) 0.01 BA (mgl-~) Explants with shoots (%) 62.51 100.0 37.5 75.0 71.4 42.9 No. of shoots per explant 2.0 6.0 0.4 2.8 1.7 0.4 0.I Explants with shoots (%) 40.0 50.0 50.0 25.0 50.0 37.5 No. of shoots per explant 0.4 0.5 2.8 1.0 1.1 0.6 0.2 Explants with shoots (%) 0 37,5 0 12.5 14.3 0 No. of shoots per explant 0 0.8 0 0.3 0.1 0 0.5 Explants with shoots (%) 0 0 0 0 0 0 No. of shoots per explant 0 0 0 0 0 0 1.0 Explants with shoots (%) 0 0 0 0 n n No. of shoots per explant 0 0 0 0 n n

1 2 3 4 5 10

~Each value represents the mean of three replicates (100 x 20 mm Petri dishes) each with 8-10 hypocotyl segments. Shoot regeneration was scored after 30 days in culture, n:not determined.

temperatures were 27 ___ 1 and 23 ___ I°C, respectively. Hypocotyls excised from just below the shoot apex of aseptically germinated seedlings were cut into upper and lower segments, each 7-8 mm long. Effect of growth regulators on shoot regeneration was investigated by transferring the upper hypocotyl segments to 100 x 20mm Petri dishes each containing 25-30ml solidified MS medium supplemented with 1, 2, 3, 4, 5 or 10mg 1-~ BA in combinations with 0.01, 0.1, 0.2, 0.5 or 1 mg 1-~ NAA. In addition, the upper segments excised from various seedling ages, e.g. 4-, 5-, 6-, 7-, 9-, 12-, 14or 18-day old, were grown on the medium containing 4mg 1-~ kinetin and 0.01 mg 1-l 2,4-D to evaluate the effect of explant age on shoot regeneration. Each treatment consisted of three replicates each with 8-10 explants. Explants were evaluated for shoot regeneration after 30 days in culture. For rooting, regenerated shoots were transferred to half-strenght growth regulator-free MS medium. The rooted shoots were transplanted into pots con-

taining soil. Plants were covered with glass beakers during the first week of acclimatization to prevent desiccation of plants.
Results

Hypocotyl explants of B. carinata grown on medium containing various combinations of BA and NAA responded differently with respect to shoot regeneration. While the highest shoot regeneration frequency (100%) accompanied by the highest number of shoots per explant (6) occurred on medium containing 2 mg 1-~ BA in combination with 0.01 mg 1 ~ NAA, regeneration was poor (050%) on BA medium containing NAA concentrations higher than 0.01 mg 1-t (Table 1). The capacity of shoot regeneration is markedly affected by the position of hypocotyl segments of which the upper segments (40.4%) was three-fold more regenerative than the lower segment (11.3%) (Table 2). In addition, the distal end of the explant was generally more regenerative, particularly for upper

Table 2. Shoot regeneration from different positions of cut end and segments of hypocotyls grown on medium containing various BA and NAA combinations.

Hypocotyl segment

No. of explants cultured

Explant producing shoots at ends of hypocotyls~ Distal No. % 39.4 5.7 Proximal No. 1 5 % 1.0 4.7 Both No. 0 1 % 0 0.9

Total shoot regeneration frequency (%)

Upper Lower

104 106

41 6

40.4 11.3

JEach value represents the sum of treatments of various BA and NAA combinations, which resulted in shoot regeneration, as described in Table I. Shoot regeneration was scored after 30 days in culture.

81
Table 3. Effect of seedling age on shoot regeneration from upper hypocotyl segments of B. carinata grown on medium containing 4 mg 1-~ kinetin and 0.01 mg 1-~ 2,4-D.

Seedling age (days) 4 5 6 7 9 12 14 18

No. of explants cultured 17 22 22 20 20 20 20 15

No. of explants producing shoots 3 15 21 20 11 13 12 9

Shoot regeneration frequency (%) 18 68 96 100 55 65 60 60

Total no. of shoots produced 10 n 78 103 15 20 20 15

No. of shoots per explant 0.6 n 3.6 5.2 0.8 1.0 1.0 1.0

Scored after 30 days in culture, n:not scored.

hypocotyl segments, than the proximal end (Table 2). While the shoot regeneration frequency of distal and proximal ends for upper segments were 39.4% and 1.0%, respectively, the respective frequency for lower segment were 5.7% and 4.7% (Table 2). Shoots rarely formed on both ends of the same explant. Although explants grown at 2 mg 1-1 BA in combination with 0.01 mg 1-1 NAA gave rise to shoots at 100% frequency, high frequency (100%) shoot regeneration also occurred for explants grown on medium containing 4 mg 1- l kinetin in combination with 0.01 mg 1 ~2,4-D (data not shown). Growth of regenerated shoots appeared to be more vigorous on kinetin/2,4-D medium than those on BA/NAA medium. Seedling age is a critical factor affecting shoot regeneration frequency. Hypocotyls derived from 6 to 7-day-old seedlings gave rise to shoots at the highest frequency (95-100%) and also produced the high number of shoots per explant (3.6-5.2). Explants originating from seedlings younger or older than 6-7 days-old resulted in a marked reduction of shoot regeneration in terms of the number of explants producing shoots and the number of shoots per explant (Table 3). Shoot cuttings originating from regenerants began to form roots after 7 days on half-strength growth regulator-free MS medium. High frequency (90-100%) rooted shoots were successfully acclimatized in the greenhouse and grew to normal, fertile plants.

Discussion

The frequency of plant regeneration from cultured cells and tissues of B. carinata was relatively low in the past [7-9]. Comparative studies on shoot regeneration response of both diploid and amphidiploid brassicas indicated that the capacity of regeneration was genetically controlled, and regenerability of B. carinata was influenced by the cytoplasmic components of its progenitors B. nigra and B. oleracea [9]. However, this study shows that shoots of B. carinata can be regenerated at high frequency (100%) via selection of explants and manipulation of culture medium. Similar findings have been obtained from B. campestris [2], for which the genome was believed to be inhibitory to regeneration [9]. The positional effect on shoot regeneration capacity is clearly demonstrated in this study, which shows higher regeneration frequency at the distal end than the proximal end of the hypocotyl explant. In addition, the upper hypocotyl segments are generally more regenerative than the lower segments. However, the mechanism of this phenomenon is not fully understood. Organogenesis is markedly affected by explant age. Previous studies have shown that shoot regeneration from cotyledons and/or hypocotyls derived from 3 to 4-day-old seedlings of B. campestris ssp. chinensis [2] and B. alboglabra [11] was substantially higher than from older seedlings. However, in this study, high frequency shoot regeneration oc-

82 curred only o n 6 to 7 - d a y - o l d h y p o c o t y l explants o f B. carinata, a n d explants y o u n g e r o r o l d e r t h a n 6 to 7 days h a d p o o r regeneration. A differential req u i r e m e n t for o p t i m a l in vitro s h o o t r e g e n e r a t i o n a m o n g Brassica species is n o t surprising since g e n o t y p e v a r i a b i l i t y for in vitro culture o f Brassica species has been well d o c u m e n t e d [4,5]. The m e t h o d described in this p a p e r might be used for in vitro selection for variants with useful characteristics, e.g. disease resistance, salt tolerance, etc. It m a y be also served as a tool for gene transfer study o f B. carinata using A g r o b a c t e r i u m t u m e f a c i e n s - m e d i a t e d t r a n s f o r m a t i o n [10].
campestris ssp. chinensis in vitro. Plant Sci. 64:243-250 3. Chuong PV, Pauls KP & BeversdorfWD (1987) Protoplast culture and plant regeneration from Brassica carinata Braun. Plant Cell Rep. 6:67-69 4. Dietert MF, Barron SA & Yoder OC (1982) Effects of genotype on in vitro culture in the genus Brassica. Plant Sci. Lett. 26:233-240 5. Jourdan PS & Earle ED (1989) Genotypic variabilityin the frequency of plant regeneration from leaf protoplasts of four Brassica spp. and Raphanus sativus. J. Am. Soc. Hortic. Sci. 114:343-349 6. Murashige T & Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15:473-497 7. Narasimhulu SB & Chopra VL (1987) Plant regeneration from callus cultures of Brassica carinata A. Br. and its implications to improvement of oil seed Brassicas. Plant Breeding 99:49-55 8. Narasimhulu SB & Chopra VL (1988) Species specific shoot regeneration response of cotyledonary explants of Brassicas. Plant Cell Rep. 7:104-106 9. Narasimhulu SB, Prakash S & Chopra VL (1988) Comparative shoot regeneration responses of diploid Brassicas and their synthetic amphidiploid products. Plant Cell Rep. 7:525-527 10. Pua E-C, Mehra-Palta A, Nagy F & Chua N-H (1987) Transgenic plants of Brassica napus L. Bio/Technology 5: 815-817 11. Pua E-C, Trinh TH & Chua N-H (1989) High frequency plant regeneration from stem explants of Brassica alboglabra Bailey in vitro. Plant Cell Tiss. Org. Cult. 17: 143152 12. Yang M-Z & Jia S-R (1989) Plant regeneration from protoplasts of Brassica carinata Braun. Acta Bot. Sin 31: 89-94

Acknowledgement
The a u t h o r s t h a n k D r Y u - Q i n g Chen, Institute o f C a s h Crops, Jiangsu A c a d e m y o f A g r i c u l t u r a l Sciences, China, for k i n d l y p r o v i d i n g the seeds o f B. carinata accession N o . 84A165.

References
1. Anand IJ, Singh JP & Malik RS (1985) B. carinata - A potential oil seed crop for rainfed agriculture. Cruciferae Newsl. 10:76-79 2. Chi G-L & Pua E-C (1989) Ethylene inhibitors enhanced de novo shoot regeneration from cotyledons of Brassica


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